Search

Development of the bioenergy sector is being actively pursued in many countries as a means to reduce climate change and fulfill international climate agreements such as the Paris Agreement. Although biomass for energy production (especially wood pellets) can replace carbon‐intensive fossil fuels, its net greenhouse gas impact varies, and the production of wood pellets can also lead to intensification in forest harvests and reduction of forest carbon stocks. Additionally, under specific conditions, emissions associated with imported biomass feedstocks may be omitted from national accounts, due to incompatibilities in accounting approaches. We assessed the risks and potential scale of emissions omitted from accounts (EOA) among key trading regions, focusing on the demand for wood pellets under different levels of climate mitigation targets. Our results suggest that the global production of wood pellets would grow from 38.9 to 120 Mton/year between 2019 and 2050 in a scenario that limits global mean temperature increase to 1.5°C above pre‐industrial levels. A large portion of this occurs in North America (36.8 Mton/year by 2050), Europe (47.6 Mton/year by 2050), and Asia (23.3 Mton/year by 2050). We estimate that in a 1.5°C scenario, global EOA associated with international trade of wood pellets has the potential to reach 23.81 MtCO2eq/year by 2030 and 69.52 MtCO2eq/year in 2050. Emissions resulting from European biomass energy production, based on wood pellet imports from the United States, may reach 11.68 MtCO2eq/year by 2030 and 33.57 MtCO2eq/year in 2050. The production of wood pellet feedstocks may also present a substantial carbon price arbitrage opportunity for bioenergy producers through a conjunction of two distinct GHG accounting rules. If this opportunity is realized, it could accelerate the growth of the bioenergy industry to levels that harm forests’ function as a carbon sink and omit actual emissions in national and global accounting frameworks.

Non-typhoidal Salmonella (NTS) serovars have a broad host range and cause gastroenteritis in humans. However, invasive NTS (iNTS) bloodstream infections have increased in the last decade, causing 60,000 deaths annually.

Forest disturbances, such as an eastern spruce budworm (Choristoneura fumiferana) outbreak, impact the strength and persistence of forest carbon sinks. Salvage harvests are a typical management response to widespread tree mortality, but the decision to salvage mortality has large implications for the fate of carbon stocks (including forest carbon and harvested wood products) in the near and long terms. In this study, we created decision-support models for salvage harvesting based on carbon after an eastern spruce budworm outbreak. We used lasso regression to determine which stand characteristics (e.g., basal area) are the best predictors of carbon 40 years after an outbreak in both salvage and no salvage scenarios. We modeled carbon at year 40 for different treatment scenarios and discount rates. Treatment scenarios represent residual stand conditions that may be present when an outbreak occurs. Economic discount rates were applied to 40-year carbon values to account for near and long-term carbon storage aspects. We found that the volume and size of eastern spruce budworm host species are significant predictors of salvage preference based on carbon. We found overall that salvaging less volume is recommended to avoid major swings in carbon budgets and that discounting carbon values to apply weight to near or long-term sequestration greatly affects whether salvaging is preferred. Lasso models are constructed for the northeastern US, however, similar concepts may be applied beyond our study area and potentially for other insect outbreaks similar to spruce budworm, such as mountain pine beetle (Dendroctonus ponderosae) or hemlock woolly adelgid (Adelges tsugae). From a policy standpoint widespread salvaging could create a large carbon emissions deficit with the risk of not being fully replenished within a desired timeframe. Since salvaging is often financially driven, especially for private landowners, carbon market payments or incentives for not salvaging is a consideration for future policy.

Nonnative invasive plant species are a major cause of ecosystem degradation and impairment of ecosystem service benefits in the United States. Forested riparian areas provide many ecosystem service benefits and are vital to maintaining water quality of streams and rivers. These systems are also vulnerable to natural disturbances and invasion by nonnative plants. We assessed whether planting native trees on disturbed riparian sites may increase biotic resistance to invasive plant establishment in central Vermont in the northeastern United States. The density (stems per square meter) of invasive stems was higher in non-planted sites (x̄ = 4.1 stems m−2) compared with planted sites (x̄ = 1.3 stems m−2). More than 90% of the invasive plants were Japanese knotweed [Fallopia japonica (Houtt.) Ronse Decr.; syn. Polygonum cuspidatum Siebold & Zucc.]. There were no significant differences in total stem density of native vegetation between planted and non-planted sites. Other measured response variables such as native tree regeneration, species diversity, soil properties, and soil function showed no significant differences or trends in the paired riparian study sites. The results of this case study indicate that tree planting in disturbed riparian forest areas may assist conservation efforts by minimizing the risk of invasive plant colonization.

Actions to protect against biodiversity loss and climate change will require a framework that addresses synergies between these interrelated issues. In this study we present methods for identifying areas important for the implementation of nature-based climate solutions and biodiversity conservation by intersecting high resolution spatial data for carbon storage and terrestrial connectivity. We explored the spatial congruence of carbon and connectivity in Ontario, Canada and examined effectiveness of current protected areas coverage. We found a weak positive relationship between carbon stocks and terrestrial connectivity; however, our maps revealed large hotspots, with high values of both indices, throughout the boreal forest and northern peatlands and smaller, isolated hotspots in the settled landscapes of the south. Location of hotspots varied depending on whether we considered forest or soil carbon. Further, our results show that current protected and conserved areas in Ontario only cover 13% of landscapes with the highest values for both carbon storage and connectivity. Protection or restoration of areas that maximize the co-benefits of carbon storage and connectivity would make significant contributions towards ambitious national targets to reduce greenhouse gas emissions and conserve biodiversity.

Typhoid fever is caused primarily by the enteric microbe Salmonella enterica serovar Typhi and remains a major global health problem with approximately 14 million new infections and 136,000 fatalities annually. While there are antibiotic options available to treat the disease, the global increase in multidrug-resistant strains necessitates alternative therapeutic options. Host-targeted therapeutics present a promising anti-infective strategy against intracellular bacterial pathogens. A cell-based assay identified a compound that inhibits

Salmonella enterica serovars cause millions of infections each year that result either in typhoid fever or salmonellosis. Among those serovars that cause typhoid fever, Salmonella enterica subspecies Typhi can form biofilms on gallstones in the gallbladders of acutely-infected patients, leading to chronic carriage of the bacterium. These biofilms are recalcitrant to antibiotic-mediated eradication, leading to chronic fecal shedding of the bacteria, which results in further disease transmission. Herein, we report the synthesis and anti-biofilm activity of a 55-member library of small molecules based upon a previously identified hit that both inhibits and disrupts S. Typhi and S. Typhimurium (a nontyphoidal model serovar for S. Typhi) biofilms. Lead compounds inhibit S. Typhimurium biofilm formation in vitro at sub-micromolar concentrations, and disperse biofilms with five-fold greater potentency than the parent compound. Three of the most promising compounds demonstrated synergy with ciprofloxacin in a murine model of chronic Salmonella carriage. This work furthers the development of effective anti-biofilm agents as a promising therapeutic avenue for the eradication of typhoidal Salmonella.

Salmonella enterica serovar Typhi causes chronic infections by establishing biofilms on cholesterol gallstones. The production of extracellular polymeric substances (EPSs) is key to biofilm development, and biofilm architecture depends on which EPSs are made. The presence and spatial distribution of Salmonella EPSs produced in vitro and in vivo were investigated in Salmonella enterica serovar Typhimurium and S. Typhi biofilms by confocal microscopy. Comparisons between serovars and EPS-mutant bacteria were carried out by examining growth on cholesterol-coated surfaces, with human gallstones in ox or human bile, and in mice with gallstones. On cholesterol-coated surfaces, no major differences in EPS biomass were found between serovars. Cocultured biofilms containing wild-type (WT) and EPS-mutant bacteria demonstrated WT compensation for EPS mutations. Analysis of biofilm EPSs from gallbladder-mimicking conditions found that culture in human bile more consistently replicated the relative abundance and spatial organization of each EPS on gallstones from the chronic mouse model than culture in ox bile. S. Typhimurium biofilms cultured in vitro on gallstones in ox bile exhibited colocalized pairings of curli fimbriae/lipopolysaccharide and O-antigen capsule/cellulose, while these associations were not present in S. Typhi biofilms or in mouse gallstone biofilms. In general, the inclusion of human bile with gallstones in vitro replicated biofilm development on gallstones in vivo, demonstrating the strength of this model for studying biofilm parameters or EPS-directed therapeutic treatments.

Knowledge of the chemical identity of metabolite molecules is critical for the understanding of the complex biological systems to which they belong. Since metabolite identities and their concentrations are often directly linked to the phenotype, such information can be used to map biochemical pathways and understand their role in health and disease. A very large number of metabolites however are still unknown; i.e., their spectroscopic signatures do not match those in existing databases, suggesting unknown molecule identification is both imperative and challenging. Although metabolites are structurally highly diverse, the majority shares a rather limited number of structural motifs, which are defined by sets of 1H and 13C chemical shifts of the same spin system. This allows one to characterize unknown metabolites by a divide-and-conquer strategy that identifies their structural motifs first. Here, we present the structural motif-based approach "SUMMIT Motif" for the de novo identification of unknown molecular structures in complex mixtures, without the need for extensive purification, using NMR in tandem with two newly curated NMR molecular structural motif metabolomics databases (MSMMDBs). For the identification of structural motif(s), first, the 1H and 13C chemical shifts of all the individual spin systems are extracted from 2D and 3D NMR spectra of the complex mixture. Next, the molecular structural motifs are identified by querying these chemical shifts against the new MSMMDBs. One database, COLMAR MSMMDB, was derived from experimental NMR chemical shifts of known metabolites taken from the COLMAR metabolomics database, while the other MSMMDB, pNMR MSMMDB, is based on predicted chemical shifts of metabolites of several existing large metabolomics databases. For molecules consisting of multiple spin systems, spin systems are connected via long-range scalar J-couplings. When this motif-based identification method was applied to the hydrophilic extract of mouse bile fluid, two unknown metabolites could be successfully identified. This approach is both accurate and efficient for the identification of unknown metabolites and hence enables the discovery of new biochemical processes and potential biomarkers.

Salmonella enterica serovar Typhi causes chronic infections by establishing biofilms on cholesterol gallstones. The production of extracellular polymeric substances (EPSs) is key to biofilm development, and biofilm architecture depends on which EPSs are made. The presence and spatial distribution of Salmonella EPSs produced in vitro and in vivo were investigated in Salmonella enterica serovar Typhimurium and S. Typhi biofilms by confocal microscopy. Comparisons between serovars and EPS-mutant bacteria were carried out by examining growth on cholesterol-coated surfaces, with human gallstones in ox or human bile, and in mice with gallstones. On cholesterol-coated surfaces, no major differences in EPS biomass were found between serovars. Cocultured biofilms containing wild-type (WT) and EPS-mutant bacteria demonstrated WT compensation for EPS mutations. Analysis of biofilm EPSs from gallbladder-mimicking conditions found that culture in human bile more consistently replicated the relative abundance and spatial organization of each EPS on gallstones from the chronic mouse model than culture in ox bile. S. Typhimurium biofilms cultured in vitro on gallstones in ox bile exhibited colocalized pairings of curli fimbriae/lipopolysaccharide and O-antigen capsule/cellulose, while these associations were not present in S. Typhi biofilms or in mouse gallstone biofilms. In general, the inclusion of human bile with gallstones in vitro replicated biofilm development on gallstones in vivo, demonstrating the strength of this model for studying biofilm parameters or EPS-directed therapeutic treatments.

L-arabinose inducible promoters are commonly used in gene expression analysis. However, nutrient source and availability also play a role in biofilm formation; therefore, L-arabinose metabolism could impact biofilm development. In this study we examined the impact of L-arabinose on Salmonella enterica serovar Typhimurium (S. Typhimurium) biofilm formation. Using mutants impaired for the transport and metabolism of L-arabinose, we showed that L-arabinose metabolism negatively impacts S. Typhimurium biofilm formation in vitro. When L-arabinose metabolism is abrogated, biofilm formation returned to baseline levels. However, without the ability to import extracellular L-arabinose, biofilm formation significantly increased. Using RNA-Seq we identified several gene families involved in these different phenotypes including curli expression, amino acid synthesis, and L-arabinose metabolism. Several individual candidate genes were tested for their involvement in the L-arabinose-mediated biofilm phenotypes, but most played no significant role. Interestingly, in the presence of L-arabinose the diguanylate cyclase gene adrA was downregulated in wild type S. Typhimurium. Meanwhile cyaA, encoding an adenylate cyclase, was downregulated in an L-arabinose transport mutant. Using an IPTG-inducible plasmid to deplete c-di-GMP via vieA expression, we were able to abolish the increased biofilm phenotype seen in the transport mutant. However, the mechanism by which the L-arabinose import mutant forms significantly larger biofilms remains to be determined. Regardless, these data suggest that L-arabinose metabolism influences intracellular c-di-GMP levels and therefore biofilm formation. These findings are important when considering the use of an L-arabinose inducible promoter in biofilm conditions.

Bacteria use two-component system (TCS) signaling pathways to sense and respond to peptides involved in host defense, quorum sensing, and inter-bacterial warfare. However, little is known about the peptide-sensing capabilities of these TCSs. Here, we develop a high-throughput E. coli display method to characterize the effects of human antimicrobial peptides (AMPs) on the pathogenesis-regulating TCS PhoPQ of Salmonella Typhimurium. We find that PhoPQ senses AMPs comprising diverse sequences, structures, and biological functions. Using thousands of AMP variants, we identify sub-domains and biophysical features responsible for PhoPQ activation. We show that most of the newfound activators induce PhoPQ in S. Typhimurium, suggesting a role in virulence regulation. Finally, we find that PhoPQ homologs from Klebsiella pneumoniae and extraintestinal pathogenic E. coli, which occupy different in vivo niches, exhibit distinct AMP response profiles. Our high-throughput method enables new insights into the specificities, mechanisms, and evolutionary dynamics of TCS-mediated peptide sensing in bacteria.

Bacteria use two-component system (TCS) signaling pathways to sense and respond to peptides involved in host defense, quorum sensing and inter-bacterial warfare. However, little is known about the broad peptide-sensing capabilities of TCSs. In this study, we developed an Escherichia coli display method to characterize the effects of human antimicrobial peptides (AMPs) on the pathogenesis-regulating TCS PhoPQ of Salmonella Typhimurium with much higher throughput than previously possible. We found that PhoPQ senses AMPs with diverse sequences, structures and biological functions. We further combined thousands of displayed AMP variants with machine learning to identify peptide sub-domains and biophysical features linked to PhoPQ activation. Most of the newfound AMP activators induce PhoPQ in S. Typhimurium, suggesting possible roles in virulence regulation. Finally, we present evidence that PhoPQ peptide-sensing specificity has evolved across commensal and pathogenic bacteria. Our method enables new insights into the specificities, mechanisms and evolutionary dynamics of TCS-mediated peptide sensing in bacteria.

Increasing demand for woody biomass-derived electricity in the UK and elsewhere has resulted in a rapidly expanding wood pellet manufacturing industry in the southern US. Since this demand is driven by climate concerns and an objective to lower greenhouse gas (GHG) emissions from the electricity sector, it is crucial to understand the full carbon consequences of wood pellet sourcing, processing, and utilization. We performed a comparative carbon life cycle assessment (LCA) for pellets sourced from three mills in the southern US destined for electricity generation in the UK. The baseline assumptions included GHG emissions of the UK’s 2018 and 2025 target electricity grid mix and feedstock supplied primarily from non-industrial private forest (NIPF) pine plantations augmented with a fraction of sawmill residues. Based on regional expert input, we concluded that forest management practices on the NIPF pine plantations would include timely thinning harvest treatments in the presence of pellet demand. The LCA analysis included landscape carbon stock changes based on USDA Forest Service Forest Vegetation Simulator using current USDA Forest Service Forest Inventory and Analysis data as the starting condition of supply areas in Arkansas, Louisiana and Mississippi. We found that GHG emission parity (i.e., the time when accumulated carbon GHG emissions for the bioenergy scenario equal the baseline scenario) is more than 40 years for pellets produced at each individual pellet mill and for all three pellet mills combined when compared to either the UK’s 2018 electricity grid mix or the UK’s targeted electricity grid mix in 2025. The urgency to mitigate climate change with near-term actions as well as increasing uncertainty with longer-term simulations dictated a focus on the next four decades in the analysis. Even at 50% sawmill residues, GHG emission parity was not reached during the 40 years modeled. Results are most likely conservative since we assume a high share of sawmill residues (ranging from 20 to 50%) and did not include limited hardwood feedstocks as reported in the supply chain which are generally associated with delayed GHG emission parity because of lower growth rates.

The ability of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S. Typhi and S. Typhimurium biofilms are tolerant to hydrogen peroxide (H2O2), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H2O2. Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S. Typhimurium and Vi antigen in S. Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H2O2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H2O2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H2O2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant (katE, katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H2O2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H2O2 because the physical barrier formed by EPSs slows penetration of H2O2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H2O2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H2O2. Thus, these data provide an explanation for how Salmonella achieves tolerance to H2O2 by a combination of an EPS-mediated barrier and enzymatic detoxification.

Sarcolipin (SLN) is a regulator of sarco/endo plasmic reticulum Ca2+-ATPase (SERCA) pump and has been shown to be involved in muscle nonshivering thermogenesis (NST) and energy metabolism. Interestingly, SLN expression is significantly upregulated both during muscle development and in several disease states. However, the significance of altered SLN expression in muscle patho-physiology is not completely understood. We have previously shown that transgenic over-expression of SLN in skeletal muscle is not detrimental, and can promote oxidative metabolism and exercise capacity. In contrast, some studies have suggested that SLN upregulation in disease states is deleterious for muscle function and ablation of SLN can be beneficial. In this perspective article, we critically examine both published and some new data to determine the relevance of SLN expression to disease pathology. The new data presented in this paper show that SLN levels are induced in muscle during systemic bacterial (Salmonella) infection or lipopolysaccharides (LPS) treatment. We also present data showing that SLN expression is significantly upregulated in different types of muscular dystrophies including myotubular myopathy. These data taken together reveal that upregulation of SLN expression in muscle disease is progressive and increases with severity. Therefore, we suggest that increased SLN expression should not be viewed as the cause of the disease; rather, it is a compensatory response to meet the higher energy demand of the muscle. We interpret that higher SLN/SERCA ratio positively modulate cytosolic Ca2+ signaling pathways to promote mitochondrial biogenesis and oxidative metabolism to meet higher energy demand in muscle.

Carbon life cycle assessments (C LCA) play a major role in greenhouse gas (GHG)-related forest management analytics for wood products and consist of several steps along a forest to disposal path. Yet, input values for wood product C LCAs frequently rely on potentially outdated generic datasets for wood product outputs and mill efficiencies. Assumptions regarding sawmill efficiencies and sawmill-specific wood product outputs have a direct and significant impact on wood product C LCAs because these variables affect the net carbon footprint of the finished product. The goal of this analysis was to evaluate how well standard wood product C LCA inputs and assumptions for the two initial wood products LCA steps (i) forest operations and (ii) wood processing represent the current state of the wood processing industry in California. We found that sawmill efficiencies and wood product outputs both support and deviate from lookup tables currently used in publications supporting the climate-forest policy dialogue. We recommend further analysis to resolve the major discrepancies in the carbon fraction stored in durable wood products and production-related emissions to improve C LCA metrics and advance forest-related climate policy discussions in California and elsewhere.

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes—an intracellular bacterial pathogen—suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host–microbial interactions. Modulation of mitochondrial metabolism can increase the survival of intracellular bacteria such as Listeria monocytogenes inside cells.

Asymptomatic carriage of Salmonella Typhi continues to facilitate the transmission of typhoid fever, resulting in 14 million new infections and 136,000 fatalities each year. Asymptomatic chronic carriage of S. Typhi is facilitated by the formation of biofilms on gallstones that protect the bacteria from environmental insults and immune system clearance. Here, we identified two unique small molecules capable of both inhibiting Salmonella biofilm growth and disrupting pre-formed biofilm structures without affecting bacterial viability. In a mouse model of chronic gallbladder Salmonella carriage, treatment with either compound reduced bacterial burden in the gallbladder by 1–2 logs resulting in bacterial dissemination to peripheral organs that was associated with increased mortality. Co-administration of either compound with ciprofloxacin not only enhanced compound efficacy in the gallbladder by a further 1–1.5 logs for a total of 3–4.5 log reduction, but also prevented bacterial dissemination to peripheral organs. These data suggest a dual-therapy approach targeting both biofilm and planktonic populations can be further developed as a safe and efficient treatment of biofilm-mediated chronic S. Typhi infections., Author summary Typhoid fever is an infectious disease caused by Salmonella Typhi (S. Typhi), a bacterium that causes as many as 14 million new infections and 136,000 deaths annually. Asymptomatic chronic carriers of S. Typhi play a major role in the transmission of typhoid fever, as they intermittently shed the bacteria and can unknowingly infect surrounding individuals. Here, we characterized novel compounds that target biofilm formation, a process utilized by S. Typhi to establish and maintain chronic carriage in the gallbladder, in hopes that they may be eventually used in conjunction with traditional antibiotics to prevent and/or cure chronic infections more efficiently than antibiotics alone.

Salmonella enterica is able to establish robust adherent communities called biofilms that allow for long-term colonization of both biotic and abiotic surfaces. These biofilm communities pose a significant challenge to successful eradication of the bacteria from contaminated surfaces and the infected host, as entry into the biofilm phenotype confers the bacterial population with tolerance to a variety of environmental and therapeutic insults to which it would otherwise be susceptible. The identification of antimicrobial strategies that specifically target the Salmonella biofilm state is therefore of great importance in order to both prevent and treat biofilm-mediated disease. Here, we provide detailed methods for the in vitro cultivation of Salmonella biofilms that can easily be scaled up for use in high-throughput screening of candidate anti-biofilm agents. These assays may also be utilized to further characterize the inhibitory and/or disruptive capabilities of lead anti-biofilm agents, as well as to identify combination treatments that demonstrate enhanced anti-biofilm effects. Furthermore, the assays may be slightly modified (e.g., optimal growth conditions) to evaluate other bacterial genera.

Salmonella enterica is able to establish robust adherent communities called biofilms that allow for long-term colonization of both biotic and abiotic surfaces. These biofilm communities pose a significant challenge to successful eradication of the bacteria from contaminated surfaces and the infected host, as entry into the biofilm phenotype confers the bacterial population with tolerance to a variety of environmental and therapeutic insults to which it would otherwise be susceptible. The identification of antimicrobial strategies that specifically target the Salmonella biofilm state is therefore of great importance in order to both prevent and treat biofilm-mediated disease. Here, we provide detailed methods for the in vitro cultivation of Salmonella biofilms that can easily be scaled up for use in high-throughput screening of candidate anti-biofilm agents. These assays may also be utilized to further characterize the inhibitory and/or disruptive capabilities of lead anti-biofilm agents, as well as to identify combination treatments that demonstrate enhanced anti-biofilm effects. Furthermore, the assays may be slightly modified (e.g., optimal growth conditions) to evaluate other bacterial genera.

The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in Francisella. IMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis. This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM. We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4(+) helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen’s ability to persist in vivo. We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4(+) helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in Francisella. IMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis. This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM. We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.