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

2021 ◽  
Vol 17 (7) ◽  
pp. e1009284
Author(s):  
Pavan Patel ◽  
Brendan J. O’Hara ◽  
Emily Aunins ◽  
Kimberly M. Davis
Keyword(s):  
Stationary Phase ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Bacterial Cells ◽  
Deep Tissue ◽  
Bacterial Populations ◽  
Novel Variant ◽  
Slow Growing ◽  
Host Tissues

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.

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

2021 ◽  
Author(s):  
Pavan Patel ◽  
Brendan J. O’Hara ◽  
Emily Aunins ◽  
Kimberly M. Davis
Keyword(s):  
Nitric Oxide ◽  
Cell Division ◽  
Stationary Phase ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Bacterial Cells ◽  
E Coli ◽  
Slow Growing ◽  
Host Tissues

AbstractIt 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.

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

2021 ◽  
Author(s):  
Bessie Liu ◽  
Robert K Davidson ◽  
Kimberly Michele Davis
Keyword(s):  
Cell Division ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Spatial Location ◽  
Mouse Spleen ◽  
Post Inoculation ◽  
Bacterial Cell Division ◽  
Bacterial Populations ◽  
Daughter Cells ◽  
Host Tissues

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.

scholarly journals For the Greater (Bacterial) Good: Heterogeneous Expression of Energetically Costly Virulence Factors

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Kimberly M. Davis
Keyword(s):  
Virulence Factor ◽  
Host Response ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Factor Expression ◽  
Heterogeneous Expression ◽  
Host Cell Interactions ◽  
Slow Growing ◽  
Kinetics Of ◽  
Host Tissues

ABSTRACT Bacterial populations are phenotypically heterogeneous, which allows subsets of cells to survive and thrive following changes in environmental conditions. For bacterial pathogens, changes within the host environment occur over the course of the immune response to infection and can result in exposure to host-derived, secreted antimicrobials or force direct interactions with immune cells. Many recent studies have shown host cell interactions promote virulence factor expression, forcing subsets of bacterial cells to battle the host response, while other bacteria reap the benefits of this pacification. It still remains unclear whether virulence factor expression is truly energetically costly within host tissues and whether expression is sufficient to impact the growth kinetics of virulence factor-expressing cells. However, it is clear that slow-growing subsets of bacteria emerge during infection and that these subsets are particularly difficult to eliminate with antibiotics. This minireview will focus on our current understanding of heterogenous virulence factor expression and discuss the evidence that supports or refutes the hypothesis that virulence factor expression is linked to slowed growth and antibiotic tolerance.

scholarly journals Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jasmine Ramirez Raneses ◽  
Alysha L. Ellison ◽  
Bessie Liu ◽  
Kimberly M. Davis
Keyword(s):  
Bacterial Growth ◽  
Intraperitoneal Injection ◽  
Bacterial Infections ◽  
Yersinia Pseudotuberculosis ◽  
Antibiotic Administration ◽  
Bacterial Cells ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Drug Concentrations ◽  
Host Tissues

ABSTRACT 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 detect exposure to doxycycline (Dox) present in 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 h after intraperitoneal injection, and by 4 h postinjection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis bacteria in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting that 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 h posttreatment. After this time point, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to that of 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. IMPORTANCE Bacterial 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 there are changes 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 we show that Dox impacts the bacterial population within hours of administration and inhibits bacterial growth for 48 h. 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 suggest that subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure.

scholarly journals Contributions ofCandida albicansDimorphism, Adhesive Interactions, and Extracellular Matrix to the Formation of Dual-Species Biofilms withStreptococcus gordonii

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Daniel Montelongo-Jauregui ◽  
Stephen P. Saville ◽  
Jose L. Lopez-Ribot
Keyword(s):  
Extracellular Matrix ◽  
Oral Cavity ◽  
Bacterial Species ◽  
Fungal Species ◽  
Bacterial Cells ◽  
Mixed Species ◽  
Bacterial Populations ◽  
Content Type ◽  
Mutant Strains ◽  
Adhesive Interactions

ABSTRACTFungal and bacterial populations coexist in the oral cavity, frequently forming mixed-species biofilms that complicate treatment against polymicrobial infections. However, despite relevance to oral health, the bidirectional interactions between these microbial populations are poorly understood. In this study, we aimed to elucidate the mechanisms underlying the interactions between the fungal speciesCandida albicansand the bacterial speciesStreptococcus gordoniias they coexist in mixed-species biofilms. Specifically, the interactions of differentC. albicansmutant strains deficient in filamentation (efg1Δ/Δ andbrg1Δ/Δ), adhesive interactions (als3Δ/Δ andbcr1Δ/Δ), and production of matrix exopolymeric substances (EPS) (kre5Δ/Δ, mnn9Δ/Δ,rlm1Δ/Δ, andzap1Δ/Δ) were evaluated withS. gordoniiunder different conditions mimicking the environment in the oral cavity. Interestingly, our results revealed that growth of the biofilm-deficientC. albicansals3Δ/Δandbcr1Δ/Δmutant strains in synthetic saliva or withS. gordoniirestored their biofilm-forming ability. Moreover, challenging previous observations indicating an important role of morphogenetic conversions in the interactions between these two species, our results indicated a highly synergistic interaction betweenS. gordoniiand theC. albicansfilamentation-deficientefg1Δ/Δandbrg1Δ/Δdeletion mutants, which was particularly noticeable when the mixed biofilms were grown in synthetic saliva. Importantly, dual-species biofilms were found to exhibit increase in antimicrobial resistance, indicating that components of the fungal exopolymeric material confer protection to streptococcal cells against antibacterial treatment. Collectively, these findings unravel a high degree of complexity in the interactions betweenC. albicansandS. gordoniiin mixed-species biofilms, which may impact homeostasis in the oral cavity.IMPORTANCEMicrobial communities have a great impact in health and disease.C. albicansinteracts with multiple microorganisms in the oral cavity, frequently forming polymicrobial biofilms. We report on the synergistic interactions betweenC. albicansand the Gram-positive bacteriumS. gordonii, for which we have examined the different contributions of adhesive interactions, filamentation, and the extracellular matrix to the formation of dual-species biofilms. Our results demonstrate that growth in the presence of the bacterium can restore the biofilm-forming ability of differentC. albicansmutant strains with defects in adhesion and filamentation. The mixed-species biofilms also show high levels of resistance to antibacterial and antifungal antibiotics, and our results indicate that the fungal biofilm matrix protects bacterial cells within these mixed-species biofilms. Our observations add to a growing body of evidence indicating a high level of complexity in the reciprocal interactions and consortial behavior of fungal/bacterial biofilms.

scholarly journals Efflux Impacts Intracellular Accumulation Only in Actively Growing Bacterial Cells

mBio ◽  
2021 ◽  
Author(s):  
Emily E. Whittle ◽  
Helen E. McNeil ◽  
Eleftheria Trampari ◽  
Mark Webber ◽  
Tim W. Overton ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Cell Envelope ◽  
Bacterial Cells ◽  
Intracellular Accumulation ◽  
Predominant Factor ◽  
Novel Drug ◽  
Intracellular Targets ◽  
Slow Growing

This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.

scholarly journals RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

2014 ◽  
Vol 80 (7) ◽  
pp. 2240-2247 ◽  
Author(s):  
Gerald W. Tannock ◽  
Blair Lawley ◽  
Karen Munro ◽  
Ian M. Sims ◽  
Julian Lee ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Bacterial Species ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Content Type ◽  
And Function

ABSTRACTKnowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed thatBacteroides uniformis,Blautia glucerasea,Clostridium indolis, andBifidobacterium animaliswere the main users of the13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.B. uniformisutilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

scholarly journals Subpopulations of stressed Y. pseudotuberculosis preferentially survive doxycycline treatment within host tissues

2020 ◽  
Author(s):  
Jasmine Ramirez Raneses ◽  
Alysha L. Ellison ◽  
Bessie Liu ◽  
Kimberly M. Davis
Keyword(s):  
Bacterial Growth ◽  
Intraperitoneal Injection ◽  
Bacterial Infections ◽  
Yersinia Pseudotuberculosis ◽  
Antibiotic Administration ◽  
Bacterial Cells ◽  
Fluorescent Reporter ◽  
Doxycycline Treatment ◽  
Drug Concentrations ◽  
Host Tissues

AbstractSevere 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.

scholarly journals Waddington’s Landscapes in the Bacterial World

2021 ◽  
Vol 12 ◽  
Author(s):  
María A. Sánchez-Romero ◽  
Josep Casadesús
Keyword(s):  
Morphological Change ◽  
Fluorescent Protein ◽  
Single Cell Analysis ◽  
Expression Patterns ◽  
Specific Gene ◽  
Bacterial Cells ◽  
Visual Metaphor ◽  
Bacterial Populations ◽  
Future Challenges ◽  
Multicellular Organisms

Conrad Waddington’s epigenetic landscape, a visual metaphor for the development of multicellular organisms, is appropriate to depict the formation of phenotypic variants of bacterial cells. Examples of bacterial differentiation that result in morphological change have been known for decades. In addition, bacterial populations contain phenotypic cell variants that lack morphological change, and the advent of fluorescent protein technology and single-cell analysis has unveiled scores of examples. Cell-specific gene expression patterns can have a random origin or arise as a programmed event. When phenotypic cell-to-cell differences are heritable, bacterial lineages are formed. The mechanisms that transmit epigenetic states to daughter cells can have strikingly different levels of complexity, from the propagation of simple feedback loops to the formation of complex DNA methylation patterns. Game theory predicts that phenotypic heterogeneity can facilitate bacterial adaptation to hostile or unpredictable environments, serving either as a division of labor or as a bet hedging that anticipates future challenges. Experimental observation confirms the existence of both types of strategies in the bacterial world.

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