scholarly journals Sulfate import in Salmonella Typhimurium impacts bacterial aggregation and the respiratory burst in human neutrophils

2021 ◽  
Author(s):  
T. L. Westerman ◽  
M. K. Sheats ◽  
J. R. Elfenbein
Keyword(s):  
Respiratory Burst ◽  
Gene Deletion ◽  
Single Gene ◽  
Protein A ◽  
Human Neutrophils ◽  
Deletion Mutants ◽  
Flagellar Motility ◽  
Neutrophil Respiratory Burst ◽  
Single Gene Deletion

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single gene deletion mutants representing particular regions under selection. A subset of single gene deletion mutants were selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

scholarly journals Sulfate import in Salmonella Typhimurium impacts bacterial aggregation and the neutrophil respiratory burst

2020 ◽  
Author(s):  
TL Westerman ◽  
MK Sheats ◽  
JR Elfenbein
Keyword(s):  
Respiratory Burst ◽  
Protein A ◽  
Flagellar Motility ◽  
Rich Media ◽  
The Individual ◽  
Genomic Regions ◽  
System 1 ◽  
Type 3 ◽  
Neutrophil Respiratory Burst

AbstractDuring enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions, followed by the individual mutants mapping to genomic regions under selection. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter neutrophil antimicrobial defenses.

scholarly journals Update on the Keio collection of Escherichia coli single‐gene deletion mutants

2009 ◽  
Vol 5 (1) ◽  
pp. 335 ◽  
Author(s):  
Natsuko Yamamoto ◽  
Kenji Nakahigashi ◽  
Tomoko Nakamichi ◽  
Mihoko Yoshino ◽  
Yuki Takai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
Keio Collection ◽  
Single Gene Deletion

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli

2020 ◽  
Author(s):  
Inés Portillo-Calderón ◽  
Miriam Ortiz-Padilla ◽  
Jose Manuel Rodríguez-Martínez ◽  
Belen de Gregorio-Iaria ◽  
Jesús Blázquez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Gene Deletion ◽  
Single Gene ◽  
Clinical Isolates ◽  
Deletion Mutants ◽  
Growth Monitoring ◽  
Fosfomycin Resistance ◽  
Time Kill ◽  
Single Gene Deletion

Abstract Objectives To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. Materials and methods We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5–512 mg/L) and time–kill assays (64 and 307 mg/L). Results Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time–kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. Conclusions The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

Behavioural phenotyping of C. elegans on UV-killed E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Uv Light ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Behavioural Phenotyping ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour when killed by ultraviolet (UV) light

scholarly journals Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils.

1992 ◽  
Vol 116 (4) ◽  
pp. 1007-1017 ◽  
Author(s):  
G Berton ◽  
C Laudanna ◽  
C Sorio ◽  
F Rossi
Keyword(s):  
Respiratory Burst ◽  
Protein A ◽  
Human Neutrophils ◽  
Necrosis Factor Alpha ◽  
Tissue Culture Polystyrene ◽  
Leukocyte Integrins ◽  
Camp Analogue ◽  
Factor Alpha ◽  
Beta 2 Microglobulin ◽  
Neutrophil Respiratory Burst

To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.

Antioxidant rescue of C. elegans behaviour on Keio E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour in the presence of antioxidants.

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