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 SdiA Aids Enterohemorrhagic Escherichia coli Carriage by Cattle Fed a Forage or Grain Diet

2013 ◽  
Vol 81 (9) ◽  
pp. 3472-3478 ◽  
Author(s):  
Haiqing Sheng ◽  
Y. N. Nguyen ◽  
Carolyn J. Hovde ◽  
Vanessa Sperandio
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Rumen Fluid ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Dependent Manner ◽  
Bacterial Survival ◽  
Wild Type

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activatedgadgene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

scholarly journals C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

2009 ◽  
Vol 8 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Alejandro D. Nusblat ◽  
Sebastián R. Najle ◽  
Mariela L. Tomazic ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
Gene Identification ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Gene Coding ◽  
Transmembrane Regions

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

Peptides’ involvement in Pseudomonas putida biofilm formation

2020 ◽  
Author(s):  
Riho Teras ◽  
Hanna Ainelo ◽  
Marge Puhm
Keyword(s):  
Pseudomonas Putida ◽  
Mutant Strain ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Positive Impact ◽  
Proteinase K ◽  
Wild Type ◽  
Positive Effect ◽  
Positively Charged

<p>Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm.</p> <p>The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N.</p> <p>We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm.</p> <p>We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain.</p> <p>In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.</p>

scholarly journals Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity

2004 ◽  
Vol 379 (3) ◽  
pp. 609-615 ◽  
Author(s):  
Daniel CLARK ◽  
Roger S. ROWLETT ◽  
John R. COLEMAN ◽  
Daniel F. KLESSIG
Keyword(s):  
Antioxidant Activity ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Carbonic Anhydrase Activity ◽  
Site Directed Mutagenesis ◽  
Carbonic Anhydrases ◽  
Wild Type ◽  
Tobacco Chloroplast ◽  
Low Levels

In recent years, members of the β class of CAs (carbonic anhydrases) have been shown to complement ΔNCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of ΔNCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196→Ala), a mutated tobacco CA with low levels of CA activity, complemented ΔNCE103. To determine whether restoration of ΔNCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs [Götz, Gnann and Zimmermann (1999) Yeast 15, 855–864], additional complementation analysis was performed using human CAII, an α CA structurally unrelated to the β class of CAs to which the tobacco protein belongs. Human CAII complemented ΔNCE103, strongly arguing that CA activity is responsible for the complementation of ΔNCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and ΔNCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.

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 Functional Characterization of Calcineurin-Responsive Transcription Factors Fg01341 and Fg01350 in Fusarium graminearum

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiangxiang Zhang ◽  
Shulin Cao ◽  
Wei Li ◽  
Haiyan Sun ◽  
Yuanyu Deng ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Functional Characterization ◽  
Hyphal Growth ◽  
Basic Medium ◽  
Dependent Manner ◽  
Wild Type ◽  
Sexual Production

Ca2 +/calmodulin-dependent phosphatase calcineurin is one of the important regulators of intracellular calcium homeostasis and has been investigated extensively in Saccharomyces cerevisiae. However, only a few reports have explored the function of the Crz1 homolog in filamentous fungi, especially in Fusarium graminearum. In this study, we identified Fg01341 as a potential ortholog of yeast Crz1. Fg01341 could interact with calcineurin and initiate nuclear transport in a calcineurin-dependent manner. The ΔFg01341 mutant exhibited normal hyphal growth on basic medium and conidia formation, but sexual reproduction was partially blocked. Pathogenicity assays showed that the virulence of the ΔFg01341 mutant in flowering wheat heads and corn silks dramatically decreased and was thus consistent with the reduction in deoxynivalenol production. Unexpectedly, the sensitivity to osmotic stress of the deletion mutant and that of the wild-type strain did not present any differences. The deletion mutant showed higher sensitivity to tebuconazole than the wild-type strain. Results also showed that the transcription factor Fg01350 might be the calcineurin target and was independent of Crz1. Furthermore, ΔFg01350 showed defects in hyphal growth, sexual production, virulence, and deoxynivalenol production. Collectively, the results indicate that these two proteins functionally redundant and that the calcineurin–Crz1-independent pathway is particularly important in F. graminearum.

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 Characterization of emeA, anorA Homolog and Multidrug Resistance Efflux Pump, inEnterococcus faecalis

2001 ◽  
Vol 45 (12) ◽  
pp. 3574-3579 ◽  
Author(s):  
Brandie M. Jonas ◽  
Barbara E. Murray ◽  
George M. Weinstock
Keyword(s):  
Drug Resistance ◽  
Multidrug Resistance ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Type Gene ◽  
Encoding Genes

ABSTRACT We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis

2015 ◽  
Vol 25 (4) ◽  
pp. 284-291
Author(s):  
Fanjie Chen ◽  
Han Hu ◽  
Zhonghua Li ◽  
Jiacheng Huang ◽  
Xuwang Cai ◽  
...  
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Sublethal Concentration ◽  
Haemophilus Parasuis ◽  
Wild Type ◽  
Microscopy Observation ◽  
Cecropin B ◽  
In The Wild ◽  
Substrate Binding Protein

Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a <i>Haemophilus parasuis</i> gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of <i>H. parasuis</i> JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 μg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to <i>H. parasuis</i> resistance to CB.

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