The involvement of lysozyme and degradation of bacterial genomic DNA in MAC-mediated killing of gram-negative bacteria

Juha Kotimaa, Sami Nikoskelainen, Hanna Jarva, Sanna Grannas, Seppo Meri

Research output: Conference materialsAbstractResearchpeer-review

Abstract

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The serum complement system and antimicrobial peptides are important elements of the humoral innate immune system that are critically involved in defence against various microbes. Tagging for recognition and uptake and disrupting the integrity of bacterial outer membranes are all well described functions of the C system. However, the downstream effects of serum leading to the killing of gram-negative bacteria with both an inner and outer membrane have been largely limited to resistance factor identification, viability analyses and monitoring growth impairment. Recent studies have established that various antibiotics trigger programmed cell death-like events in gram-negative bacteria, including rapid degradation of genomic DNA. We hypothesized that membrane disruption by serum complement, specific antibiotics affecting the membrane and anti-microbial proteins could lead to a similar effect. We employed standard agarose gel-electrophoresis, loss of 3H-thymidine incorporated DNA and flow cytometric analysis of TUNEL staining to assess the integrity of the bacterial genomic DNA after exposure to normal human serum. All three methods showed degradation of DNA within 2 h of NHS treatment, with concomitant loss of viability and membrane integrity. In addition to being complement-dependent, we identified the need for serum lysozyme based on its ability to promote DNA degradation in bacteria, and further showed a dose-dependent abrogation of DNA fragmentation when lysozyme was neutralized. Further research showed that the serum-killing effect was similar to polymyxin B, a membrane disrupting antibiotic. Interestingly, this effect seems to be dependent on bacterial processes, as NHS without addition of exogenous DNA had no DNA degrading effect on paraffin fixed bacteria. The established effect of complement and polymyxin B is their ability to disrupt membrane potential and function, which are vital for the bacterial homeostasis. Recent studies have shown that lysozyme may deposit to bacterial membranes and convey similar membrane disrupting effect that is likely to be cumulative with complement and polymyxin B. We hypothesize that this disruption leads to stress responses that eventually escalate to bacterial programmed cell death via activation of bacterial endonucleases. Host innate immune cells could subsequently be activated by the bacterial DNA fragments.
Original languageEnglish
Pages176-176
Number of pages1
Publication statusPublished - Oct 2018
MoE publication typeNot Eligible

Cite this

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title = "The involvement of lysozyme and degradation of bacterial genomic DNA in MAC-mediated killing of gram-negative bacteria",
abstract = "Previous articleNext articleThe serum complement system and antimicrobial peptides are important elements of the humoral innate immune system that are critically involved in defence against various microbes. Tagging for recognition and uptake and disrupting the integrity of bacterial outer membranes are all well described functions of the C system. However, the downstream effects of serum leading to the killing of gram-negative bacteria with both an inner and outer membrane have been largely limited to resistance factor identification, viability analyses and monitoring growth impairment. Recent studies have established that various antibiotics trigger programmed cell death-like events in gram-negative bacteria, including rapid degradation of genomic DNA. We hypothesized that membrane disruption by serum complement, specific antibiotics affecting the membrane and anti-microbial proteins could lead to a similar effect. We employed standard agarose gel-electrophoresis, loss of 3H-thymidine incorporated DNA and flow cytometric analysis of TUNEL staining to assess the integrity of the bacterial genomic DNA after exposure to normal human serum. All three methods showed degradation of DNA within 2 h of NHS treatment, with concomitant loss of viability and membrane integrity. In addition to being complement-dependent, we identified the need for serum lysozyme based on its ability to promote DNA degradation in bacteria, and further showed a dose-dependent abrogation of DNA fragmentation when lysozyme was neutralized. Further research showed that the serum-killing effect was similar to polymyxin B, a membrane disrupting antibiotic. Interestingly, this effect seems to be dependent on bacterial processes, as NHS without addition of exogenous DNA had no DNA degrading effect on paraffin fixed bacteria. The established effect of complement and polymyxin B is their ability to disrupt membrane potential and function, which are vital for the bacterial homeostasis. Recent studies have shown that lysozyme may deposit to bacterial membranes and convey similar membrane disrupting effect that is likely to be cumulative with complement and polymyxin B. We hypothesize that this disruption leads to stress responses that eventually escalate to bacterial programmed cell death via activation of bacterial endonucleases. Host innate immune cells could subsequently be activated by the bacterial DNA fragments.",
author = "Juha Kotimaa and Sami Nikoskelainen and Hanna Jarva and Sanna Grannas and Seppo Meri",
year = "2018",
month = "10",
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The involvement of lysozyme and degradation of bacterial genomic DNA in MAC-mediated killing of gram-negative bacteria. / Kotimaa, Juha; Nikoskelainen, Sami; Jarva, Hanna; Grannas, Sanna; Meri, Seppo .

2018. 176-176.

Research output: Conference materialsAbstractResearchpeer-review

TY - CONF

T1 - The involvement of lysozyme and degradation of bacterial genomic DNA in MAC-mediated killing of gram-negative bacteria

AU - Kotimaa, Juha

AU - Nikoskelainen, Sami

AU - Jarva, Hanna

AU - Grannas, Sanna

AU - Meri, Seppo

PY - 2018/10

Y1 - 2018/10

N2 - Previous articleNext articleThe serum complement system and antimicrobial peptides are important elements of the humoral innate immune system that are critically involved in defence against various microbes. Tagging for recognition and uptake and disrupting the integrity of bacterial outer membranes are all well described functions of the C system. However, the downstream effects of serum leading to the killing of gram-negative bacteria with both an inner and outer membrane have been largely limited to resistance factor identification, viability analyses and monitoring growth impairment. Recent studies have established that various antibiotics trigger programmed cell death-like events in gram-negative bacteria, including rapid degradation of genomic DNA. We hypothesized that membrane disruption by serum complement, specific antibiotics affecting the membrane and anti-microbial proteins could lead to a similar effect. We employed standard agarose gel-electrophoresis, loss of 3H-thymidine incorporated DNA and flow cytometric analysis of TUNEL staining to assess the integrity of the bacterial genomic DNA after exposure to normal human serum. All three methods showed degradation of DNA within 2 h of NHS treatment, with concomitant loss of viability and membrane integrity. In addition to being complement-dependent, we identified the need for serum lysozyme based on its ability to promote DNA degradation in bacteria, and further showed a dose-dependent abrogation of DNA fragmentation when lysozyme was neutralized. Further research showed that the serum-killing effect was similar to polymyxin B, a membrane disrupting antibiotic. Interestingly, this effect seems to be dependent on bacterial processes, as NHS without addition of exogenous DNA had no DNA degrading effect on paraffin fixed bacteria. The established effect of complement and polymyxin B is their ability to disrupt membrane potential and function, which are vital for the bacterial homeostasis. Recent studies have shown that lysozyme may deposit to bacterial membranes and convey similar membrane disrupting effect that is likely to be cumulative with complement and polymyxin B. We hypothesize that this disruption leads to stress responses that eventually escalate to bacterial programmed cell death via activation of bacterial endonucleases. Host innate immune cells could subsequently be activated by the bacterial DNA fragments.

AB - Previous articleNext articleThe serum complement system and antimicrobial peptides are important elements of the humoral innate immune system that are critically involved in defence against various microbes. Tagging for recognition and uptake and disrupting the integrity of bacterial outer membranes are all well described functions of the C system. However, the downstream effects of serum leading to the killing of gram-negative bacteria with both an inner and outer membrane have been largely limited to resistance factor identification, viability analyses and monitoring growth impairment. Recent studies have established that various antibiotics trigger programmed cell death-like events in gram-negative bacteria, including rapid degradation of genomic DNA. We hypothesized that membrane disruption by serum complement, specific antibiotics affecting the membrane and anti-microbial proteins could lead to a similar effect. We employed standard agarose gel-electrophoresis, loss of 3H-thymidine incorporated DNA and flow cytometric analysis of TUNEL staining to assess the integrity of the bacterial genomic DNA after exposure to normal human serum. All three methods showed degradation of DNA within 2 h of NHS treatment, with concomitant loss of viability and membrane integrity. In addition to being complement-dependent, we identified the need for serum lysozyme based on its ability to promote DNA degradation in bacteria, and further showed a dose-dependent abrogation of DNA fragmentation when lysozyme was neutralized. Further research showed that the serum-killing effect was similar to polymyxin B, a membrane disrupting antibiotic. Interestingly, this effect seems to be dependent on bacterial processes, as NHS without addition of exogenous DNA had no DNA degrading effect on paraffin fixed bacteria. The established effect of complement and polymyxin B is their ability to disrupt membrane potential and function, which are vital for the bacterial homeostasis. Recent studies have shown that lysozyme may deposit to bacterial membranes and convey similar membrane disrupting effect that is likely to be cumulative with complement and polymyxin B. We hypothesize that this disruption leads to stress responses that eventually escalate to bacterial programmed cell death via activation of bacterial endonucleases. Host innate immune cells could subsequently be activated by the bacterial DNA fragments.

M3 - Abstract

SP - 176

EP - 176

ER -