Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism

Jones-Carson, Jessica; Yahashiri, Atsushi; Kim, Ju-Sim; Liu, Lin; Fitzsimmons, Liam F.; Weiss, David S.; Vázquez‐Torres, Andrés

doi:10.1126/sciadv.aaz0260

“…These results provide additional insight into the mechanism of NO-mediated stress, and suggest the response to NO is sufficient to arrest cell division, but not promote a stationary phase transition. This is consistent with published results showing NO disrupts cell division by arresting assembly of the FtsZ cytokinetic ring [46,47].…”

Section: Dsred 42 Signal Accumulation Correlates With Hmp Expression During Exposure To Acidified Nitritesupporting

confidence: 93%

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

Patel

¹

,

O’Hara

²

,

Aunins

³

et al. 2021

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It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.

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“…Our in vitro results were consistent with this, where exposure to NO was sufficient for TIMER42 signal accumulation. Our results are also consistent with a recently published study which showed that NO causes a collapse of proton motive force (PMF) and collapse of the divisome complex, thus resulting in cell division arrest (39). However, within host tissues, slow-growing bacterial cells with TIMER42 signal accumulation were largely Hmp -, and instead expressed the stationary phase marker, Dps.…”

Section: Discussionsupporting

confidence: 92%

“…These results provide additional insight into the mechanism of NO-mediated stress, and suggest the response to NO is sufficient to arrest cell division, but not promote a stationary phase transition. This is consistent with published results showing NO disrupts cell division by arresting assembly of the FtsZ cytokinetic ring (38,39).…”

Section: No Stress Is Sufficient To Promote Timer42 Signal Accumulationsupporting

confidence: 93%

“…NO exposure has recently been shown to arrest bacterial cell division through disruption of cytokinesis machinery, and so it was expected that TIMER42 signal would accumulate in growth-arrested, NO-exposed, bacteria (39). The lack of correlation between the two signals during growth in tissue was unexpected, and suggested that NO stress may have occurred much earlier during infection than the 72h timepoint.…”

Section: Timer42 Signal Dissipates After Initial Responses To No Sugmentioning

confidence: 99%

See 1 more Smart Citation

Modifying TIMER, a slow-folding DsRed derivative, for optimal use in quickly-dividing bacteria

Patel

¹

,

O’Hara

²

,

Aunins

³

et al. 2021

Preprint

2

0

View full text Add to dashboard Cite

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however this TIMER42 variant accumulates signal in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show TIMER42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting TIMER42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, TIMER42 signal is clearly detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was not significant overlap between TIMER42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel TIMER42 variant represents a new faster folding TIMER that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.Author SummaryWe have generated a variant of TIMER that can be used to mark slow-growing subsets of Yersinia pseudotuberculosis, which has a relatively short division time, similar to E. coli. We used a combination of site-directed and random mutagenesis to generate the TIMER42 variant, which has red fluorescent signal accumulation in post-exponential or stationary phase cells. We found that nitric oxide (NO) stress is sufficient to promote TIMER42 signal accumulation in culture, however within host tissues, TIMER42 signal correlates with a stationary phase reporter (dps). These results suggest NO may cause an immediate arrest in bacterial cell division, but during growth in host tissues exposure to NO is transient, allowing bacteria to recover from this stress and resume cell division. Thus instead of indicating a response to host stressors, TIMER42 signal accumulation within host tissues appears to identify slow-growing cells that are experiencing nutrient limitation.

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“…6D). Similar to the uncoupler CCCP, NO also depolarizes the cytoplasmic membrane to arrest respiration and growth [48, 49]. Consistent with this, and as seen with CCCP, pretreatment with DETA-NO also increased basal OCR of wt Mtb and Mtb Δ sufR (Fig.…”

Section: Resultssupporting

confidence: 65%

Mycobacterium tuberculosisSufR Responds to Nitric oxide via its 4Fe-4S cluster and Regulates Fe-S cluster Biogenesis for Persistence in Mice

Anand

¹

,

Tripathi

²

,

Shukla

³

et al. 2020

Preprint

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Host-inducible nitric oxide (NO) suppresses Mycobacterium tuberculosis (Mtb) without eradicating it. Mechanisms underlying the persistence of residual bacteria in a hostile host environment are not known. Since NO damages Fe-S clusters of essential enzymes, the mechanism used to repair Fe-S clusters could help Mtb persist in host tissues. Here, we show that Mtb utilizes the suf operon (Rv1460-Rv1466) for Fe-S cluster biogenesis to persist under NO stress. Further, transcriptional repressor SufR (Rv1460) senses NO through its 4Fe-4S cluster to directly regulate Suf-meditated Fe-S cluster biogenesis. We discovered that NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory reserves and redox buffering capacity of MtbΔsufR. Importantly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics of Mtb.

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Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism

Cited by 31 publications

References 63 publications

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

Modifying TIMER, a slow-folding DsRed derivative, for optimal use in quickly-dividing bacteria

Mycobacterium tuberculosisSufR Responds to Nitric oxide via its 4Fe-4S cluster and Regulates Fe-S cluster Biogenesis for Persistence in Mice

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