Iron-sulfur cluster repair contributes to <i>Y. pseudotuberculosis</i> survival within deep tissues

Davis, Kimberly M.; Krupp, Joanna; Clark, Stacie; Isberg, Ralph R.

doi:10.1101/576454

Cited by 3 publications

(11 citation statements)

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“…Bacteria at the periphery of the microcolony respond to NO by expressing the NO-detoxifying gene, hmp (18). hmp expression at the periphery prevents NO diffusion into the center of microcolonies, establishing an example of cooperative behavior, where the peripheral bacteria protect the interior bacteria from the antimicrobial action of NO (18, 19). It remains unclear if the protective expression of hmp by peripheral cells comes at a fitness cost, and if this stress response is sufficient to alter the antibiotic susceptibility of this subpopulation.…”

Section: Introductionmentioning

confidence: 99%

Subpopulations of stressedY. pseudotuberculosispreferentially survive doxycycline treatment within host tissues

Raneses

Ellison

Liu

et al. 2020

Preprint

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Severe systemic bacterial infections result in colonization of deep tissues, which can be very difficult to eliminate with antibiotics. It remains unclear if this is because antibiotics are not reaching inhibitory concentrations within tissues, if subsets of bacteria are less susceptible to antibiotics, or if both contribute to limited treatment efficacy. To determine the concentration of doxycycline (Dox) present within deep tissues following treatment, we generated a fluorescent transcriptional reporter derived from the tet operon to specifically detect intracellular tetracycline exposure at the single bacterial cell level. Dox exposure was detected in the spleen 2 hours after intraperitoneal injection, and by 4 hours post-injection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting stress was sufficient to alter Dox susceptibility. Many bacteria (~10%) survived a single dose of Dox, and the antibiotic accumulated at the periphery of microcolonies to growth inhibitory concentrations until 48 hours post-treatment. After this timepoint, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to untreated mice. These results indicate that Dox delivery by intraperitoneal injection results in rapid diffusion of inhibitory concentrations of antibiotic into the spleen, but stressed cells preferentially survive drug treatment, and bacterial growth resumes once drug concentrations decrease. This fluorescent reporter strategy for antibiotic detection could easily be modified to detect the concentration of additional antimicrobial compounds within host tissues following drug administration.ImportanceBacterial infections are very difficult to treat when bacteria spread into the bloodstream and begin to replicate within deep tissues, such as the spleen. Subsets of bacteria can survive antibiotic treatment, but it remains unclear if this survival is because of limited drug diffusion into tissues, or if something has changed within the bacteria, promoting survival of some bacterial cells. Here, we have developed a fluorescent reporter to detect doxycycline (Dox) diffusion into host tissues, and show that Dox impacts the bacterial population within hours of administration, and inhibits bacterial growth for 48 hours. However, bacterial growth resumes when antibiotic concentrations decrease. Subsets of bacteria, stressed by the host response to infection, survive Dox treatment at a higher rate. These results provide critical information about the dynamics that occur within deep tissues following antibiotic administration, and suggests subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure.

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Section: Introductionmentioning

confidence: 99%

Subpopulations of stressedY. pseudotuberculosispreferentially survive doxycycline treatment within host tissues

Raneses

Ellison

Liu

et al. 2020

Preprint

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“…We then sought to move TIMER42 into the low copy vector pMMB67EH, which is stably maintained during mouse infection (22,26). Insertion of TIMER42 into the pMMB67EH multiple cloning site (MCS) did not result in detectable fluorescence despite the upstream Ptac promoter, so we also inserted PhmsT, the promoter for the diguanylate cyclase hmsT, upstream of TIMER42.…”

Section: Timer42mentioning

confidence: 99%

“…To quantify the relative TIMER signal accumulation within individual bacterial cells, we transformed TIMER42-expressing Yptb (PhmsT::TIMER42) with a plasmid that constitutivelyexpresses GFP (22,26). To determine when TIMER42 signal accumulated during bacterial growth in the spleen, we intravenously infected C57BL/6 mice with this GFP + TIMER42 strain and harvested spleens at 48h and 72h post-inoculation (p.i.)…”

Section: Timer42 Is Detected Within the Host Spleen At 72h Pimentioning

confidence: 99%

“…We then sought to determine if the response to reactive nitrogen species (RNS) is sufficient to slow the growth of cells and promote TIMER signal accumulation, since we observe bacterial responses to this stress during growth in the spleen (22,26). To determine if RNS were sufficient to promote TIMER42 signal accumulation, we first used the acidified nitrite method of generating nitric oxide (NO), where NaNO2 is added to acidified LB (pH 5.5) (22,26,36). The 2.5mM NaNO2 concentration was known to inhibit growth and promote high levels of hmp expression, whereas 0.25mM represents a sub-inhibitory concentration (26).…”

Section: Timer42 Signal Accumulation Correlates With Hmp Expression Dmentioning

confidence: 99%

“…Bacteria at the periphery of microcolonies respond to neutrophil contact by expressing high levels of the anti-phagocytic type-III secretion system and its associated effector proteins (22). Peripheral bacteria also respond to and detoxify the diffusible antimicrobial gas, nitric oxide (NO), which is produced by recruited monocytes circumscribing the neutrophil layer (22,26). NO detoxification occurs through expression of hmp, a nitric oxide dioxygenase, which converts antimicrobial NO to the innocuous NO3 (27).…”

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

Patel

O’Hara

Aunins

et al. 2021

Preprint

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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, 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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NO-stressed Y. pseudotuberculosis have decreased cell division rates in the mouse spleen

Liu

Davidson

Davis

2021

Preprint

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Fluorescence dilution approaches can detect bacterial cell division events, and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein, and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated a revTetR reporter construct, where mCherry is constitutively expressed, and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined timeframes. We show that mCherry signal is diluted in replicating populations, and that mCherry signal accumulates in growth-inhibited populations, including during exposure to inhibitory concentrations of antibiotics and during nitric oxide exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection. We defined a drug concentration that results in even exposure of cells to tetracyclines, and used this system to visualize cell division within defined timeframes post-inoculation. revTetR mCherry signal did not appear enriched in a particular spatial location within replicating centers of bacteria. However, the addition of a NO-sensing reporter (Phmp::gfp) showed that heightened NO exposure correlated with heightened mCherry signal, suggesting decreased cell division within this subpopulation. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.

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Iron-sulfur cluster repair contributes to Y. pseudotuberculosis survival within deep tissues

Cited by 3 publications

References 50 publications

Subpopulations of stressedY. pseudotuberculosispreferentially survive doxycycline treatment within host tissues

Subpopulations of stressedY. pseudotuberculosispreferentially survive doxycycline treatment within host tissues

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

NO-stressed Y. pseudotuberculosis have decreased cell division rates in the mouse spleen

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