scholarly journals AcpA Is a Francisella Acid Phosphatase That Affects Intramacrophage Survival and Virulence

2006 ◽  
Vol 75 (1) ◽  
pp. 390-396 ◽  
Author(s):  
Nrusingh P. Mohapatra ◽  
Ashwin Balagopal ◽  
Shilpa Soni ◽  
Larry S. Schlesinger ◽  
John S. Gunn
Keyword(s):  
Acid Phosphatase ◽  
Intracellular Trafficking ◽  
Type Strain ◽  
Wild Type Strain ◽  
Human Monocyte ◽  
Human Macrophage ◽  
Wild Type ◽  
Intracellular Killing ◽  
Francisella Novicida ◽  
Transmission Electron

ABSTRACT 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.

scholarly journals Roles of Capsule and Lipopolysaccharide O Antigen in Interactions of Human Monocyte-Derived Dendritic Cells and Klebsiella pneumoniae

2009 ◽  
Vol 78 (1) ◽  
pp. 210-219 ◽  
Author(s):  
B. Evrard ◽  
D. Balestrino ◽  
A. Dosgilbert ◽  
J.-L. J. Bouya-Gachancard ◽  
N. Charbonnel ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Klebsiella Pneumoniae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cytokine Production ◽  
Human Monocyte ◽  
Confocal Laser ◽  
Wild Type ◽  
O Antigen ◽  
Th1 Cytokine

ABSTRACT In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

scholarly journals Shigella flexneri IpaH7.8 Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages

2000 ◽  
Vol 68 (6) ◽  
pp. 3608-3619 ◽  
Author(s):  
Carmen M. Fernandez-Prada ◽  
David L. Hoover ◽  
Ben D. Tall ◽  
Antoinette B. Hartman ◽  
June Kopelowitz ◽  
...  
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Shigella Flexneri ◽  
Human Monocyte ◽  
Avirulent Strain ◽  
Human Monocytes ◽  
Wild Type ◽  
Distribution Profile ◽  
J774 Cells

ABSTRACT The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH 7.8 deletion mutant ofS. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting thatipaH 7.8 deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressingipaH 7.8 restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH 4.5 oripaH 9.8, however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH 7.8 gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

scholarly journals A Second Two-Component Regulatory System of Bordetella bronchiseptica Required for Bacterial Resistance to Oxidative Stress, Production of Acid Phosphatase, and In Vivo Persistence

1998 ◽  
Vol 66 (10) ◽  
pp. 4640-4650 ◽  
Author(s):  
Heidrun Jungnitz ◽  
Nicholas P. West ◽  
Mark J. Walker ◽  
Gursharan S. Chhatwal ◽  
Carlos A. Guzmán
Keyword(s):  
Oxidative Stress ◽  
Acid Phosphatase ◽  
Type Strain ◽  
Bacterial Resistance ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Bordetella Bronchiseptica ◽  
Mammalian Host ◽  
Wild Type ◽  
Two Component

ABSTRACT Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetellaspp., the bvg locus. A risA′-′lacZtranslational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of β-galactosidase activity under different environmental conditions suggested that ris is regulated independently ofbvg and is optimally expressed at 37°C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetellaspp., is required for the expression of acid phosphatase byB. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive andbvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.

scholarly journals Vaccination with an Attenuated Strain of Francisella novicida Prevents T-Cell Depletion and Protects Mice Infected with the Wild-Type Strain from Severe Sepsis

2009 ◽  
Vol 77 (10) ◽  
pp. 4314-4326 ◽  
Author(s):  
Jyotika Sharma ◽  
Qun Li ◽  
Bibhuti B. Mishra ◽  
Michelle J. Georges ◽  
Judy M. Teale
Keyword(s):  
Severe Sepsis ◽  
T Cell ◽  
Type Strain ◽  
Wild Type Strain ◽  
Severe Pneumonia ◽  
Cell Depletion ◽  
Lung Pathology ◽  
Wild Type ◽  
T Cell Depletion ◽  
Francisella Novicida

ABSTRACT Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of αβ T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.

scholarly journals Protein Arginine N-methyltransferases 5 and 7 Promote HIV-1 Production

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 355 ◽  
Author(s):  
Hironobu Murakami ◽  
Takehiro Suzuki ◽  
Kiyoto Tsuchiya ◽  
Hiroyuki Gatanaga ◽  
Manabu Taura ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Virus Production ◽  
Human Monocyte ◽  
Wild Type ◽  
Viral Reservoirs ◽  
Immunodeficiency Virus ◽  
Current Therapies ◽  
Hiv 1

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.

Identification and characterization of a polysaccharide deacetylase gene from Bacillus thuringiensis

2006 ◽  
Vol 52 (10) ◽  
pp. 935-941 ◽  
Author(s):  
Kun Hu ◽  
Haihua Yang ◽  
Gang Liu ◽  
Huarong Tan
Keyword(s):  
Bacillus Thuringiensis ◽  
Spore Germination ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Disruption Mutant ◽  
Transmission Electron ◽  
Significant Difference ◽  
Polymerase Chain

One polysaccharide deacetylase gene was cloned from Bacillus thuringiensis and designated pdaA. Disruption of pdaA did not affect vegetative growth and sporulation but obviously affected spore germination. When L-alanine was added into the spore suspension, the spores of the pdaA disruption mutant showed a slow and partial reduction in absorbance at OD600 and became phase pale gray compared with phase dark of the wild-type strain. In contrast with the outgrowing of wild-type spores after germination, the pdaA mutant spores were blocked at the stage of spore germination. Transmission electron micrographs revealed a significant difference between the pdaA mutant and the wild-type strain in the spore cortex. Introduction of the pdaA gene into the pdaA disruption mutant complemented the germination-negative phenotype. Reverse transcription – polymerase chain reaction showed that pdaA was transcribed after incubation for 10 h in CCY medium.Key words: Bacillus thuringiensis, polysaccharide deacetylase, spore germination, microscopy.

scholarly journals Acquisition of the Vacuolar ATPase Proton Pump and Phagosome Acidification Are Essential for Escape of Francisella tularensis into the Macrophage Cytosol

2008 ◽  
Vol 76 (6) ◽  
pp. 2671-2677 ◽  
Author(s):  
Marina Santic ◽  
Rexford Asare ◽  
Ivana Skrobonja ◽  
Snake Jones ◽  
Yousef Abu Kwaik
Keyword(s):  
Francisella Tularensis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Human Monocyte ◽  
Vacuolar Atpase ◽  
Specific Inhibition ◽  
Bafilomycin A1 ◽  
Wild Type ◽  
Bacterial Proliferation ◽  
Acidic Compartments

ABSTRACT The Francisella tularensis-containing phagosome (FCP) matures to a late-endosome-like phagosome prior to bacterial escape into the cytosols of macrophages, where bacterial proliferation occurs. Our data show that within the first 15 min after infection of primary human monocyte-derived macrophages (hMDMs), ∼90% of the FCPs acquire the proton vacuolar ATPase (vATPase) pump and the lysomotropic dye LysoTracker, which concentrates in acidic compartments, similar to phagosomes harboring the Listeria monocytogenes control. The acquired proton vATPase pump and lysomotropic dye are gradually lost by 30 to 60 min postinfection, which coincides with bacterial escape into the cytosols of hMDMs. Colocalization of phagosomes harboring the iglD mutant with the vATPase pump and the LysoTracker dye was also transient, and the loss of colocalization was faster than that observed for the wild-type strain, which is consistent with the faster escape of the iglD mutant into the macrophage cytosol. In contrast, colocalization of both makers with phagosomes harboring the iglC mutant was persistent, which is consistent with fusion to the lysosomes and failure of the iglC mutant to escape into the macrophage cytosol. We have utilized a fluorescence microscopy-based phagosome integrity assay for differential labeling of vacuolar versus cytosolic bacteria, using antibacterial antibodies loaded into the cytosols of live hMDMs. We show that specific inhibition of the proton vATPase pump by bafilomycin A1 (BFA) blocks rapid bacterial escape into the cytosols of hMDMs, but 30% to 50% of the bacteria escape into the cytosol by 6 to 12 h after BFA treatment. The effect of BFA on the blocking of bacterial escape into the cytosol is completely reversible, as the bacteria escape after removal of BFA. We also show that the limited fusion of the FCP to lysosomes is not due to failure to recruit the late-endosomal fusion regulator Rab7. Therefore, within few minutes of its biogenesis, the FCP transiently acquires the proton vATPase pump to acidify the phagosome, and this transient acidification is essential for subsequent bacterial escape into the macrophage cytosol.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wei Liu ◽  
Xue Bai ◽  
Yan Li ◽  
Haikun Zhang ◽  
Xiaoke Hu
Keyword(s):  
Signal Transduction ◽  
Host Plant ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Azorhizobium Caulinodans ◽  
Wild Type ◽  
Response Regulators ◽  
Free Living ◽  
Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

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