scholarly journals BPSS1504, a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

2014 ◽  
Vol 82 (5) ◽  
pp. 2006-2015 ◽  
Author(s):  
Verena Hopf ◽  
André Göhler ◽  
Kristin Eske-Pogodda ◽  
Antje Bast ◽  
Ivo Steinmetz ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Burkholderia Pseudomallei ◽  
Plaque Assay ◽  
Giant Cells ◽  
Host Cells ◽  
Wild Type ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTBurkholderia pseudomalleiis a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide.B. pseudomalleiharbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified aB. pseudomalleitransposon mutant defective in theBPSS1504gene that showed reduced plaque formation. TheBPSS1504locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis ofB. pseudomalleiin mammalian hosts. For further analysis, aB. pseudomalleiBPSS1504deletion (BpΔBPSS1504) mutant and complemented mutant strain were constructed.B. pseudomalleilacking theBPSS1504gene was highly attenuated in BALB/c mice, whereas thein vivovirulence of the complemented mutant strain was fully restored to the wild-type level. TheBpΔBPSS1504mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lackinghcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS componentsvgrG,tssA, andhcp1, as well as the T6SS regulatorsvirAG,bprC, andbsaN, was not dependent onBPSS1504expression. However, secretion of Hcp1 was not detectable in the absence ofBPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

scholarly journals Regulation of Type VI Secretion System during Burkholderia pseudomallei Infection

2011 ◽  
Vol 79 (8) ◽  
pp. 3064-3073 ◽  
Author(s):  
Yahua Chen ◽  
Jocelyn Wong ◽  
Guang Wen Sun ◽  
Yichun Liu ◽  
Gek-Yen Gladys Tan ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Host Cells ◽  
Virulence Determinants ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Content Type ◽  
Regulatory Cascade ◽  
Type Vi Secretion ◽  
Absolute Dependence

ABSTRACTType III and type VI secretion systems (T3SSs and T6SSs, respectively) are critical virulence determinants in several Gram-negative pathogens. InBurkholderia pseudomallei, the T3SS-3 and T6SS-1 clusters have been implicated in bacterial virulence in mammalian hosts. We recently discovered a regulatory cascade that coordinately controls the expression of T3SS-3 and T6SS-1. BsaN is a central regulator located within T3SS-3 for the expression of T3SS-3 effectors and regulators for T6SS-1 such as VirA-VirG (VirAG) and BprC. Whereas T6SS-1 gene expression was completely dependent on BprC when bacteria were grown in medium, the expression inside host cells was dependent on the two-component sensor-regulator VirAG, with the exception of thetssABoperon, which was dependent primarily on BprC. VirAG and BprC initiate different transcriptional start sites within T6SS-1, and VirAG is able to activate thehcp1promoter directly. We also provided novel evidence thatvirAG,bprC, andtssABare critical for T6SS-1 function in macrophages. Furthermore,virAGandbprCregulator mutants were avirulent in mice, demonstrating the absolute dependence of T6SS-1 expression on these regulatorsin vivo.

scholarly journals Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Shawn M. Zimmerman ◽  
Jeremy S. Dyke ◽  
Tomislav P. Jelesijevic ◽  
Frank Michel ◽  
Eric R. Lafontaine ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Protective Immunity ◽  
Burkholderia Pseudomallei ◽  
Lipolytic Activity ◽  
Burkholderia Mallei ◽  
Wild Type ◽  
Content Type ◽  
Related Organism ◽  
Genes Encoding

ABSTRACT Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei. Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo. Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

scholarly journals Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

2015 ◽  
Vol 84 (3) ◽  
pp. 701-710 ◽  
Author(s):  
Madeleine G. Moule ◽  
Natasha Spink ◽  
Sam Willcocks ◽  
Jiali Lim ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Wild Type ◽  
Content Type ◽  
Growth And Survival ◽  
New Genes ◽  
Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

scholarly journals Two Functional Type VI Secretion Systems in Avian Pathogenic Escherichia coli Are Involved in Different Pathogenic Pathways

2014 ◽  
Vol 82 (9) ◽  
pp. 3867-3879 ◽  
Author(s):  
Jiale Ma ◽  
Yinli Bao ◽  
Min Sun ◽  
Wenyang Dong ◽  
Zihao Pan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chicken Embryo Fibroblast ◽  
Microvascular Endothelial Cell ◽  
Infection Model ◽  
Tail Fiber ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTType VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenicEscherichia coli(APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucialclpVclusters of these two T6SS loci and theirvgrGgenes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines culturedin vitro, decreased pathogenicity in duck and mouse infection modelsin vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.

scholarly journals The Burkholderia pseudomallei Enoyl-Acyl Carrier Protein Reductase FabI1 Is Essential forIn VivoGrowth and Is the Target of a Novel Chemotherapeutic with Efficacy

2013 ◽  
Vol 58 (2) ◽  
pp. 931-935 ◽  
Author(s):  
Jason E. Cummings ◽  
Luke C. Kingry ◽  
Drew A. Rholl ◽  
Herbert P. Schweizer ◽  
Peter J. Tonge ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Specific Inhibitor ◽  
Carrier Protein ◽  
Wild Type ◽  
Content Type ◽  
Fatty Acid Biosynthesis Pathway ◽  
Enoyl Acp Reductase

ABSTRACTThe bacterial fatty acid biosynthesis pathway is a validated target for the development of novel chemotherapeutics. However, sinceBurkholderia pseudomalleicarries genes that encode both FabI and FabV enoyl-acyl carrier protein (ACP) reductase homologues, the enoyl-ACP reductase that is essential forin vivogrowth needs to be defined so that the correct drug target can be chosen for development. Accordingly, ΔfabI1, ΔfabI2, and ΔfabVknockout strains were constructed and tested in a mouse model of infection. Mice infected with a ΔfabI1strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ΔfabI2and ΔfabVmutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ΔfabI2and ΔfabVstrains were not significantly different from those of the wild-type strain, a slight delay was observed. A FabI1-specific inhibitor was used to confirm that inhibition of FabI1 results in reduced bacterial burden and efficacy in an acuteB. pseudomalleimurine model of infection. This work establishes that FabI1 is required for growth ofBurkholderia pseudomalleiin vivoand is a potential molecular target for drug development.

scholarly journals A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

2011 ◽  
Vol 79 (9) ◽  
pp. 3665-3676 ◽  
Author(s):  
Brian C. Russo ◽  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Deanna M. Schmitt ◽  
Matthew J. Brown ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Febrile Illness ◽  
Host Cells ◽  
High Morbidity ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Schu S4

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agentFrancisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by whichF. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions ofF. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis ofF. tularensissubsp.holarcticalive vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 inF. tularensissubsp.tularensisSchu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophagesin vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuatedin vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as anF. tularensisgene important for virulence and evasion of the host immune response.

scholarly journals Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

2013 ◽  
Vol 81 (4) ◽  
pp. 1334-1340 ◽  
Author(s):  
Nelly Leung ◽  
Antonella Gianfelice ◽  
Scott D. Gray-Owen ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

scholarly journals Identification of Burkholderia pseudomallei Genes Required for the Intracellular Life Cycle and In Vivo Virulence

2006 ◽  
Vol 74 (6) ◽  
pp. 3576-3586 ◽  
Author(s):  
Sabine Pilatz ◽  
Katrin Breitbach ◽  
Nadine Hein ◽  
Beate Fehlhaber ◽  
Jessika Schulze ◽  
...  
Keyword(s):  
Life Cycle ◽  
Burkholderia Pseudomallei ◽  
Plaque Assay ◽  
Bacterial Pathogen ◽  
Hypothetical Protein ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Endocytic Vesicles

ABSTRACT The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

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