ABSTRACT
The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in FrancisellaIMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.
Subject(s)
Bacterial Proteins/metabolism , Francisella/enzymology , Histidine Kinase/metabolism , Membrane Proteins/metabolism , Virulence Factors/metabolism , Animals , Cell Line , Francisella/pathogenicity , Gene Expression Regulation, Bacterial , Macrophages/microbiology , Mice , VirulenceABSTRACT
Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47 (phox) and replicated in macrophages isolated from p47 (phox) knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.
Subject(s)
Acid Phosphatase/metabolism , Francisella tularensis/enzymology , Francisella tularensis/physiology , Acid Phosphatase/deficiency , Acid Phosphatase/genetics , Animals , Female , Francisella tularensis/genetics , Gene Deletion , Humans , Macrophages/cytology , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Monocytes/cytology , Monocytes/microbiology , NADPH Oxidases/metabolism , Neutrophils/cytology , Neutrophils/microbiology , Phagosomes/microbiology , Protein Transport , Reactive Oxygen Species/metabolismABSTRACT
Francisella tularensis is a remarkably infectious facultative intracellular pathogen that causes the zoonotic disease tularemia. Essential to the pathogenesis of F. tularensis is its ability to escape the destructive phagosomal environment and inhibit the host cell respiratory burst. F. tularensis subspecies encode a series of acid phosphatases, which have been reported to play important roles in Francisella phagosomal escape, inhibition of the respiratory burst, and intracellular survival. However, rigorous demonstration of acid phosphatase secretion by intracellular Francisella has not been shown. Here, we demonstrate that AcpA, which contributes most of the F. tularensis acid phosphatase activity, is secreted into the culture supernatant in vitro by F. novicida and F. tularensis subsp. holarctica LVS. In addition, both F. novicida and the highly virulent F. tularensis subsp. tularensis Schu S4 strain are able to secrete and also translocate AcpA into the host macrophage cytosol. This is the first evidence of acid phosphatase translocation during macrophage infection, and this knowledge will greatly enhance our understanding of the functions of these enzymes in Francisella pathogenesis.
Subject(s)
Acid Phosphatase/metabolism , Francisella tularensis/enzymology , Francisella tularensis/pathogenicity , Macrophages/microbiology , Virulence Factors/metabolism , Animals , Cells, Cultured , Humans , Protein TransportABSTRACT
Francisella tularensis subsp. tularensis is the etiologic agent of tularemia and has been designated a category A biothreat agent by the CDC. Tularemia is characterized by replication and dissemination within host phagocytes. Intramacrophage growth is dependent upon the regulation of Francisella pathogenicity island (FPI) virulence genes, which is poorly understood. Two-component regulatory systems (TCS) are widely employed by Gram-negative bacteria to monitor and respond to environmental signals. Virulent strains of F. tularensis subsp. tularensis are devoid of classical, tandemly arranged TCS genes, but orphaned members, such as that encoding the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant shows decreased expression of FPI genes, is deficient for intramacrophage growth, and is avirulent in the mouse model. Here, we determine that phosphorylation aids PmrA binding to regulated promoters pmrA and the FPI-encoded pdpD, and KdpD is the histidine kinase primarily responsible for phosphorylation of PmrA at the aspartic acid at position 51 (D51). A strain expressing PmrA D51A retains some DNA binding but exhibits reduced expression of the PmrA regulon, is deficient for intramacrophage replication, and is attenuated in the mouse model. With regard to virulence gene induction, PmrA coprecipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together, these data suggest a model of Francisella gene regulation that includes a TCS consisting of KdpD and PmrA. Once phosphorylated, PmrA binds to regulated gene promoters recruiting free or RNA polymerase-bound MglA and SspA to initiate FPI gene transcription.
Subject(s)
Bacterial Proteins/metabolism , Francisella/pathogenicity , Gene Expression Regulation, Bacterial , Protein Kinases/metabolism , Virulence Factors/biosynthesis , Adhesins, Bacterial/metabolism , Amino Acid Substitution , Animals , Bacterial Proteins/genetics , Female , Immunoprecipitation , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Microbial Viability , Mutagenesis, Site-Directed , Phosphorylation , Protein Binding , Protein Interaction Mapping , Survival Analysis , Tularemia/microbiology , VirulenceABSTRACT
Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.
Subject(s)
Acid Phosphatase/physiology , Francisella tularensis/enzymology , Francisella tularensis/immunology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Phagocytes/enzymology , Phagocytes/microbiology , Acid Phosphatase/genetics , Animals , Cells, Cultured , Francisella tularensis/growth & development , Humans , Intracellular Fluid/enzymology , Intracellular Fluid/immunology , Intracellular Fluid/microbiology , Isoenzymes/genetics , Isoenzymes/physiology , Macrophages/enzymology , Macrophages/immunology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/biosynthesis , Neutrophils/enzymology , Neutrophils/immunology , Neutrophils/microbiology , Phagocytes/immunology , Phosphorylation/immunology , Reactive Oxygen Species/metabolism , Respiratory Burst/immunologyABSTRACT
Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD(50) in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious.
ABSTRACT
Francisella tularensis is one of the most virulent bacteria known and a Centers for Disease Control and Prevention Category A select agent. It is able to infect a variety of animals and insects and can persist in the environment, thus Francisella spp. must be able to survive in diverse environmental niches. However, F. tularensis has a surprising dearth of sensory and regulatory factors. Recent advancements in the field have identified new functions of encoded transcription factors and greatly expanded our understanding of virulence gene regulation. Here we review the current knowledge of environmental adaptation by F. tularensis, its transcriptional regulators and their relationship to animal virulence.
ABSTRACT
Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.
Subject(s)
Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Bacterial Proteins/antagonists & inhibitors , Francisella tularensis/enzymology , Macrophages/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain/physiology , Francisella tularensis/genetics , Gene Deletion , Macrophages/microbiology , Mice , Microbial Viability/drug effects , Microbial Viability/genetics , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolismABSTRACT
BACKGROUND: Francisella tularensis is a gram-negative coccobacillus that causes the febrile illness tularemia. Subspecies that are pathogenic for humans include those comprising the type A (subspecies tularensis) or type B (subspecies holarctica) biovars. An attenuated live vaccine strain (LVS) developed from a type B isolate has previously been used to vaccinate at-risk individuals, but offers limited protection against high dose (>1000 CFUs) challenge with type A strains delivered by the respiratory route. Due to differences between type A and type B F. tularensis strains at the genetic level, it has been speculated that utilization of an attenuated type A strain as a live vaccine might offer better protection against homologous respiratory challenge compared with LVS. Here, we report the construction and characterization of an unmarked Delta purMCD mutant in the highly virulent type A strain Schu S4. METHODOLOGY/PRINCIPAL FINDINGS: Growth of Schu S4 Delta purMCD was severely attenuated in primary human peripheral blood monocyte-derived macrophages and in the A549 human lung epithelial cell line. The Schu S4 Delta purMCD mutant was also highly attenuated in mice when delivered via either the intranasal or intradermal infection route. Mice vaccinated intranasally with Schu S4 Delta purMCD were well protected against high dose intradermal challenge with virulent type A or type B strains of F. tularensis. However, intranasal vaccination with Schu S4 Delta purMCD induced tissue damage in the lungs, and conferred only limited protection against high dose Schu S4 challenge delivered by the same route. The level of protection observed was similar to that conferred following vaccination with wild-type LVS or the analogous LVS Delta purMCD mutant. CONCLUSIONS/SIGNIFICANCE: Collectively, these results argue that development of the next generation live attenuated vaccine for Francisella should be based on use of the less pathogenic type B biovar rather than the more reactogenic type A biovar.
Subject(s)
Bacterial Vaccines/immunology , Francisella tularensis/immunology , Purines/immunology , Administration, Intranasal , Animals , Bacterial Vaccines/administration & dosage , Cell Line , Francisella tularensis/genetics , Francisella tularensis/pathogenicity , Humans , Lung/microbiology , Macrophages/microbiology , Mice , Mutation , VirulenceABSTRACT
Francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. It is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. Bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. The goal of this study was to delete the four primary acid phosphatases (Acps) from Francisella novicida and examine the interactions of mutant strains with macrophages, as well as the virulence of these strains in mice. We constructed F. novicida mutants with various combinations of acp deletions and showed that loss of the four Acps (AcpA, AcpB, AcpC, and histidine acid phosphatase [Hap]) in an F. novicida strain (DeltaABCH) resulted in a 90% reduction in acid phosphatase activity. The DeltaABCH mutant was defective for survival/growth within human and murine macrophage cell lines and was unable to escape from phagosome vacuoles. With accumulation of Acp deletions, a progressive loss of virulence in the mouse model was observed. The DeltaABCH strain was dramatically attenuated and was an effective single-dose vaccine against homologous challenge. Furthermore, both acpA and hap were induced when the bacteria were within host macrophages. Thus, the Francisella acid phosphatases cumulatively play an important role in intracellular trafficking and virulence.
Subject(s)
Acid Phosphatase/genetics , Bacterial Proteins/genetics , Francisella tularensis/enzymology , Francisella tularensis/pathogenicity , Macrophages/microbiology , Phagosomes/microbiology , Virulence Factors/genetics , Animals , Cell Line , Colony Count, Microbial , Female , Francisella tularensis/genetics , Francisella tularensis/growth & development , Gene Deletion , Gene Order , Humans , Liver/microbiology , Mice , Mice, Inbred BALB C , Spleen/microbiology , Survival Analysis , VirulenceABSTRACT
Francisella tularensis is a category A agent of biowarfare/biodefense. Little is known about the regulation of virulence gene expression in Francisella spp. Comparatively few regulatory factors exist in Francisella, including those belonging to two-component systems (TCS). However, orphan members of typical TCS can be identified. To determine if orphan TCS members affect Francisella gene expression, a gene encoding a product with high similarity to the Salmonella PmrA response regulator (FTT1557c/FNU0663.2) was deleted in Francisella novicida (a model organism for F. tularensis). The F. novicida pmrA mutant was defective in survival/growth within human and murine macrophage cell lines and was 100% defective in virulence in mice at a dose of up to 10(8) CFU. In addition, the mutant strain demonstrated increased susceptibility to antimicrobial peptide killing, but no differences were observed between the lipid A of the mutant and the parental strain, as has been observed with pmrA mutants of other microbes. The F. novicida pmrA mutant was 100% protective as a single-dose vaccine when challenge was with 10(6) CFU of F. novicida but did not protect against type A Schu S4 wild-type challenge. DNA microarray analysis identified 65 genes regulated by PmrA. The majority of these genes were located in the region surrounding pmrA or within the Francisella pathogenicity island (FPI). These FPI genes are also regulated by MglA, but MglA does not regulate pmrA, nor does PmrA regulate MglA. Thus, the orphan response regulator PmrA is an important factor in controlling virulence in F. novicida, and a pmrA mutant strain is an effective vaccine against homologous challenge.
Subject(s)
Bacterial Proteins/metabolism , Francisella/pathogenicity , Gene Expression Regulation, Bacterial , Genomic Islands/genetics , Signal Transduction , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Vaccines/administration & dosage , Cell Line , Female , Francisella/genetics , Francisella/immunology , Francisella/metabolism , Gene Expression Profiling , Genomic Islands/physiology , Gram-Negative Bacterial Infections/prevention & control , Humans , Macrophages , Mice , Mice, Inbred BALB C , Mutation , Oligonucleotide Array Sequence Analysis , VirulenceABSTRACT
AcpA of Francisella spp. is a respiratory-burst-inhibiting acid phosphatase that also exhibits phospholipase C activity. To better understand the molecular basis of AcpA in virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant compared to the wild type and were found mostly associated with the outer membrane. The acpA mutant was more susceptible to intracellular killing than the wild-type strain in the THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant survived longer than the wild-type strain and were less fit than the wild-type strain in competition infection assays. Transmission electron microscopy showed that the acpA mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA mutant bacteria could still be found inside phagosomes after 12 h of infection in THP-1 cells and human monocyte-derived macrophages, whereas most of the wild-type bacteria had escaped from the phagosome by 6 h postinfection. Thus, AcpA affects intracellular trafficking and the fate of Francisella within host macrophages.
Subject(s)
Acid Phosphatase/immunology , Bacterial Proteins/immunology , Francisella/pathogenicity , Macrophages/immunology , Macrophages/microbiology , Acid Phosphatase/metabolism , Animals , Bacterial Proteins/metabolism , Cell Survival , Female , Francisella/enzymology , Humans , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Phagosomes/immunology , Phagosomes/microbiology , Polymerase Chain Reaction , VirulenceABSTRACT
Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.