Suppression of ERK activation in urethral epithelial cells infected with Neisseria gonorrhoeae and its isogenic minD mutant contributes to anti-apoptosis

Liu, Guanqun; Parti, Rajinder P.; Dillon, Jo-Anne R.

doi:10.1016/j.micinf.2014.12.012

Cited by 4 publications

(2 citation statements)

References 18 publications

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“…Adherence of N. gonorrhoeae to ME-180 cells was determined by seeding 2 × 10 5 ME-180 cells/well in a 24 well tissue culture plate (Costar #3524, Corning Incorporated, Corning, USA) for 18–20 h at 80–90% confluency in antibiotic-free RPMI medium. ME-180 cells were infected with 200 μl (2 × 10 7 cfu/ml) of CFSE-stained N. gonorrhoeae F62 at a multiplicity of infection (MOI) of 1:100 and incubated for 2 h in a humidified chamber, with 5% ( v /v) CO 2 at 37 °C [18]. ME-180 cells alone and ME-180 cells infected with non-stained bacteria were used as negative controls.…”

Section: Methodsmentioning

confidence: 99%

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

et al. 2019

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BackgroundNeisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5′-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells.ResultsWe observed that 20.3% (+/− 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner.ConclusionsFlow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.Electronic supplementary materialThe online version of this article (10.1186/s12866-019-1438-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

et al. 2019

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“…In a separate instance, intracellular gonococci reduced ERK activation in transduced human urethral epithelial cells, which was correlated to a reduction in apoptosis. However, how this ERK-reduction leads to differences concerning the mitochondrial-mediated pathway was not investigated (Liu et al, 2015).…”

Section: Apoptosismentioning

confidence: 99%

Mechanisms of host manipulation by Neisseria gonorrhoeae

et al. 2023

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Neisseria gonorrhoeae (also known as gonococcus) has been causing gonorrhoea in humans since ancient Egyptian times. Today, global gonorrhoea infections are rising at an alarming rate, in concert with an increasing number of antimicrobial-resistant strains. The gonococcus has concurrently evolved several intricate mechanisms that promote pathogenesis by evading both host immunity and defeating common therapeutic interventions. Central to these adaptations is the ability of the gonococcus to manipulate various host microenvironments upon infection. For example, the gonococcus can survive within neutrophils through direct regulation of both the oxidative burst response and maturation of the phagosome; a concerning trait given the important role neutrophils have in defending against invading pathogens. Hence, a detailed understanding of how N. gonorrhoeae exploits the human host to establish and maintain infection is crucial for combating this pathogen. This review summarizes the mechanisms behind host manipulation, with a central focus on the exploitation of host epithelial cell signaling to promote colonization and invasion of the epithelial lining, the modulation of the host immune response to evade both innate and adaptive defenses, and the manipulation of host cell death pathways to both assist colonization and combat antimicrobial activities of innate immune cells. Collectively, these pathways act in concert to enable N. gonorrhoeae to colonize and invade a wide array of host tissues, both establishing and disseminating gonococcal infection.

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ERK1/2 and the Bcl-2 Family Proteins Mcl-1, tBid, and Bim Are Involved in Inhibition of Apoptosis During Persistent Chlamydia psittaci Infection

Wang

Wen

et al. 2018

Inflammation

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Chlamydia psittaci is an obligate intracellular pathogen that can cause zoonosis. Persistent C. psittaci infection can inhibit apoptosis in host cells, thus extending their survival and enabling them to complete their growth cycle. In this study, the antiapoptotic effects of persistent C. psittaci infection, induced by treatment with IFN-γ, were found to be associated with both the death receptor and the mitochondrial pathways of apoptosis. These effects were mediated by Bcl-2 family members, as evidenced by the decreased expression of proapoptotic proteins, such as tBid and Bim. Simultaneously, the antiapoptotic protein Mcl-1 was upregulated by persistent C. psittaci infection. Increased phosphorylation of ERK1/2 was observed; however, the expression of Bad, unlike that of other proapoptotic proteins, did not seem to be involved in this process. In summary, persistent chlamydial infection exerts antiapoptotic effects through both the death receptor and the mitochondrial pathways, in a process that is regulated by the ERK1/2 and apoptotic proteins of the Bcl-2 family.

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Suppression of ERK activation in urethral epithelial cells infected with Neisseria gonorrhoeae and its isogenic minD mutant contributes to anti-apoptosis

Cited by 4 publications

References 18 publications

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester

Mechanisms of host manipulation by Neisseria gonorrhoeae

ERK1/2 and the Bcl-2 Family Proteins Mcl-1, tBid, and Bim Are Involved in Inhibition of Apoptosis During Persistent Chlamydia psittaci Infection

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