scholarly journals A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1498-1504 ◽  
Author(s):  
Crystal L. Schmerk ◽  
Barry N. Duplantis ◽  
Perry L. Howard ◽  
Francis E. Nano
Keyword(s):  
Growth Factors ◽  
Francisella Tularensis ◽  
Transcriptional Response ◽  
Wild Type ◽  
Intracellular Replication ◽  
Intracellular Growth ◽  
Francisella Novicida ◽  
Mrna Profile ◽  
Growth Phenotype ◽  
In Cis

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the ΔpdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

scholarly journals Effects of the Putative Transcriptional Regulator IclR on Francisella tularensis Pathogenesis

2010 ◽  
Vol 78 (12) ◽  
pp. 5022-5032 ◽  
Author(s):  
Brittany L. Mortensen ◽  
James R. Fuller ◽  
Sharon Taft-Benz ◽  
Todd M. Kijek ◽  
Cheryl N. Miller ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Transcriptional Regulator ◽  
Live Vaccine Strain ◽  
Negative Bacterium ◽  
Wild Type ◽  
Intracellular Growth ◽  
Intranasal Inoculation ◽  
Number Of Genes ◽  
The Impact ◽  
Highly Virulent

ABSTRACT Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 ΔiclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔiclR were equally cytotoxic and induced equivalent levels of interleukin-1β expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and ΔiclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.

scholarly journals MglA and Igl Proteins Contribute to the Modulation of Francisella tularensis Live Vaccine Strain-Containing Phagosomes in Murine Macrophages

2008 ◽  
Vol 76 (8) ◽  
pp. 3502-3510 ◽  
Author(s):  
Linda Bönquist ◽  
Helena Lindgren ◽  
Igor Golovliov ◽  
Tina Guina ◽  
Anders Sjöstedt
Keyword(s):  
Cell Line ◽  
Francisella Tularensis ◽  
Vaccine Strain ◽  
Live Vaccine ◽  
Live Vaccine Strain ◽  
Intracellular Replication ◽  
Global Regulator ◽  
Intracellular Growth ◽  
Impaired Ability ◽  
Macrophagelike Cell

ABSTRACT The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.

scholarly journals Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity

2020 ◽  
Vol 10 ◽  
Author(s):  
Nasibeh Mohammadi ◽  
Helena Lindgren ◽  
Igor Golovliov ◽  
Kjell Eneslätt ◽  
Masahiro Yamamoto ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Culture System ◽  
Dominant Role ◽  
Severe Disease ◽  
Effective Control ◽  
Wild Type ◽  
Intracellular Replication ◽  
Immune Mouse ◽  
Schu S4

Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBPchr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs.

scholarly journals Tryptophan Prototrophy Contributes to Francisella tularensis Evasion of Gamma Interferon-Mediated Host Defense

2011 ◽  
Vol 79 (6) ◽  
pp. 2356-2361 ◽  
Author(s):  
Ping Chu ◽  
Annette R. Rodriguez ◽  
Bernard P. Arulanandam ◽  
Karl E. Klose
Keyword(s):  
Francisella Tularensis ◽  
Host Defense ◽  
Defined Medium ◽  
Gamma Interferon ◽  
Wild Type ◽  
Intracellular Growth ◽  
Content Type ◽  
Interferon Receptor ◽  
High Virulence ◽  
Ifn Γ

ABSTRACTFrancisella tularensisis able to survive and replicate within host macrophages, a trait that is associated with the high virulence of this bacterium. ThetrpABgenes encode the enzymes required for the final two steps in tryptophan biosynthesis, with TrpB being responsible for the conversion of indole to tryptophan. Consistent with this function, anF. tularensissubsp.novicidatrpBmutant is unable to grow in defined medium in the absence of tryptophan. ThetrpBmutant is also attenuated for virulence in a mouse pulmonary model of tularemia. However, thetrpBmutant remains virulent in gamma interferon receptor-deficient (IFN-γR−/−) mice, demonstrating that IFN-γ-mediated signaling contributes to clearance of thetrpBmutant. IFN-γ limits intracellular survival of thetrpBmutant within bone marrow-derived macrophages from wild-type but not IFN-γR−/−mice. AnF. tularensissubsp.tularensis trpBmutant is also attenuated for virulence in mice and survival within IFN-γ-treated macrophages, indicating that tryptophan prototrophy is also important in a human-virulentF. tularensissubspecies. These results demonstrate thattrpBcontributes toF. tularensisvirulence by enabling intracellular growth under IFN-γ-mediated tryptophan limitation.

scholarly journals Francisella tularensis subsp. tularensis Schu S4 Disulfide Bond Formation Protein B, but Not an RND-Type Efflux Pump, Is Required for Virulence

2008 ◽  
Vol 76 (7) ◽  
pp. 3086-3092 ◽  
Author(s):  
Aiping Qin ◽  
David W. Scott ◽  
Barbara J. Mann
Keyword(s):  
Francisella Tularensis ◽  
Disulfide Bond ◽  
Efflux Pump ◽  
Bond Formation ◽  
Sublethal Dose ◽  
Disulfide Bond Formation ◽  
Wild Type ◽  
Intracellular Growth ◽  
Schu S4

ABSTRACT Francisella tularensis subsp. tularensis is a highly virulent bacterium that is a CDC select agent. Despite advancements in the understanding of its biology, details pertaining to virulence are poorly understood. In previous work, we identified a transposon insertion mutant in the FTT0107c locus that was defective in intracellular survival in HepG2 and J77A.1 cells. Here, we report that this mutant was also highly attenuated in vivo. The FTT0107c locus is predicted to encode an ortholog of the disulfide bond formation B protein (DsbB). This designation was confirmed by complementation of an Escherichia coli dsbB mutant. This dsbB mutant of Schu S4 was highly attenuated in mice, but unlike what has been reported for Francisella novicida, intranasal immunization with a sublethal dose did not induce protection against wild-type challenge. dsbB was found to be transcribed in an operon with acrA and acrB, which encode an RND-type efflux pump. However, this pump did not make a significant contribution to virulence because strains with nonpolar deletions in acrA and acrB behaved like wild-type strain Schu S4 with respect to intracellular growth and in vivo virulence. This result is in contrast to a report that an acrB mutant of a live vaccine strain of F. tularensis has decreased virulence in mice. Overall, these results demonstrate key differences between the virulence requirements of Schu S4 and less virulent subspecies of Francisella. We have shown that DsbB is a key participant in intracellular growth and virulence, and our results suggest that there are critical virulence factors that contain disulfide bonds.

scholarly journals Multiple T Cell Subsets Control Francisella tularensis LVS Intracellular Growth Without Stimulation Through Macrophage Interferon γ Receptors

2003 ◽  
Vol 198 (3) ◽  
pp. 379-389 ◽  
Author(s):  
Siobhán C. Cowley ◽  
Karen L. Elkins
Keyword(s):  
T Cells ◽  
T Cell ◽  
Francisella Tularensis ◽  
T Cell Subsets ◽  
Wild Type ◽  
Intracellular Growth ◽  
Tnf Α ◽  
Ifn Γ

A variety of data suggest that in vivo production of interferon (IFN)-γ is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-γ–independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow–derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-γ receptor knockout (IFNγR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNγR KO macrophages. Further, a unique population of Thy1+αβ+CD4−CD8− cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNγR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) α. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-γ receptor; such control is due in large part to TNF-α, and is partially mediated by a unique double negative T cell subpopulation.

scholarly journals Perforin- and Granzyme-Mediated Cytotoxic Effector Functions Are Essential for Protection against Francisella tularensis following Vaccination by the Defined F. tularensis subsp. novicida ΔfopCVaccine Strain

2012 ◽  
Vol 80 (6) ◽  
pp. 2177-2185 ◽  
Author(s):  
Shilpa Sanapala ◽  
Jieh-Juen Yu ◽  
Ashlesh K. Murthy ◽  
Weidang Li ◽  
M. Neal Guentzel ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Nk Cell ◽  
Immune Serum ◽  
Wild Type ◽  
Vaccine Platform ◽  
Class I Mhc ◽  
Mhc I ◽  
Content Type ◽  
Francisella Novicida ◽  
The Difference

ABSTRACTA licensed vaccine againstFrancisella tularensisis currently not available. TwoFrancisella tularensissubsp.novicida(herein referred to by its earlier name,Francisella novicida) attenuated strains, the ΔiglBand ΔfopCstrains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals.F. novicidaΔiglB, aFrancisellapathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereasF. novicidaΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of theF. novicidaΔiglBand ΔfopCmutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However,F. novicidaΔfopC-vaccinated but notF. novicidaΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicidaΔfopCimmune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) inF. novicidaΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuatedF. novicidastrains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.

scholarly journals Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 321-326 ◽  
Author(s):  
H Mitsuzawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Slow Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Triple Mutant ◽  
Apparent Absence ◽  
Camp Pathway ◽  
Naturally Occurring ◽  
Elevated Activity ◽  
Growth Phenotype

Abstract The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

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