Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into ...glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD+ and by SpxB and PDHc to generate acetyl‐CoA. In galactose, pyruvate metabolism was shunted toward acetyl‐CoA production. The majority of acetyl‐CoA generated by PFL was used to regenerate NAD+ with a subset used in capsule production, while the acetyl‐CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.
Virulence of Streptococcus pneumoniae is modulated by balance of carbohydrate metabolism via pyruvate node enzymes, providing a means to pass through the mucin layer and gain access past the epithelial layer. Loss of this balance results in changes in the pyruvate flux, levels of key metabolites, capsule production, and capacity to invade and survive in the host.
Streptococcus pneumoniae is an opportunistic pathogen that colonizes the upper respiratory tract asymptomatically and, upon invasion, can lead to severe diseases including otitis media, sinusitis, ...meningitis, bacteremia, and pneumonia. One of the first lines of defense against pneumococcal invasive disease is inflammation, including the recruitment of neutrophils to the site of infection. The invasive pneumococcus can be cleared through the action of serine proteases generated by neutrophils. It is less clear how serine proteases impact non-invasive pneumococcal colonization, which is the key first step to invasion and transmission. One significant aspect of pneumococcal biology and adaptation in the respiratory tract is its natural competence, which is triggered by a small peptide CSP. In this study, we investigate if serine proteases are capable of degrading CSP and the impact this has on pneumococcal competence. We found that CSP has several potential sites for trypsin-like serine protease degradation and that there were preferential cleavage sites recognized by the proteases. Digestion of CSP with two different trypsin-like serine proteases dramatically reduced competence in a dose-dependent manner. Incubation of CSP with mouse lung homogenate also reduced recombination frequency of the pneumococcus. These ex vivo experiments suggested that serine proteases in the lower respiratory tract reduce pneumococcal competence. This was subsequently confirmed measuring in vivo recombination frequencies after induction of protease production via poly (I:C) stimulation and via co-infection with influenza A virus, which dramatically lowered recombination events. These data shed light on a new mechanism by which the host can modulate pneumococcal behavior and genetic exchange via direct degradation of the competence signaling peptide.
The capacity of Streptococcus pneumoniae to successfully transmit and colonize new human hosts is a critical aspect of pneumococcal population biology and a prerequisite for invasive disease. ...However, the bacterial mechanisms underlying this process remain largely unknown. To identify bacterial factors required for transmission, we conducted a high-throughput genetic screen with a transposon sequencing (Tn-seq) library of a pneumococcal strain in a ferret transmission model. Key players in both metabolism and transcriptional regulation were identified as required for efficient bacterial transmission. Targeted deletion of the putative C3-degrading protease CppA, iron transporter PiaA, or competence regulatory histidine kinase ComD significantly decreased transmissibility in a mouse model, further validating the screen. Maternal vaccination with recombinant surface-exposed PiaA and CppA alone or in combination blocked transmission in offspring and were more effective than capsule-based vaccines. These data underscore the possibility of targeting pneumococcal transmission as a means of eliminating invasive disease in the population.
Display omitted
•A pneumococcal Tn-seq library was screened in a ferret transmission model•The fitness landscape of S. pneumoniae genes during mammalian transmission established•Metabolic factors enhance pneumococcal environmental stability•Vaccinating dams with identified factors blocks pneumococcal transmission in offspring
Rowe et al. conduct a screen to identify pneumococcal genes required for effective transmission in a ferret model. They establish the fitness landscape of S. pneumoniae genes during mammalian transmission and find that maternal vaccination with the identified transmission factors can block bacterial transmission in the offspring.
is the causative agent of a multitude of diseases, and further study into its pathogenies is vital. The pneumococcus is genetically malleable, and several tools are available to manipulate this ...pathogen. In this study, we attempted to utilize one such tool, the Sweet Janus cassette, to replace the capsule locus with other capsule loci in our strain background and found that the efficiency of allelic replacement was low and the number of revertant false-positive colonies was high. We determined that the capacity to recombine capsule varied by the initial isolated colony, suggesting that frequency of reversion is dependent on the bacterial clone. Alternative selection markers may further expand the application of Sweet Janus. We created novel cassettes that utilized chlorinated phenylalanine as an alternative counter-selection agent in conjunction with the Janus or Sweet Janus cassette, providing a new dual or triple selection marker. Moreover, we created cassettes that do not require engineered resistance in the background strain, including both single and dual selection markers. We were able to utilize all constructs in allelic replacement of the capsule loci. These novel constructs provide a new means for generating gene deletions in
that expand experimental applications.
Efficient and highly organized regulation of transcription is fundamental to an organism's ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of ...transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5'-untranslated regions (5'-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5'-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.
Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the ...transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by
S. pneumoniae
coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.
ABSTRACT
Influenza A virus (IAV) is a major pathogen of the human respiratory tract, where the virus coexists and interacts with bacterial populations comprising the respiratory tract microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these virus-bacterium interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic-treated ferrets by coinfection with
Streptococcus pneumoniae
confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.
IMPORTANCE
Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by
S. pneumoniae
coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.
is a major human pathogen of global health concern and the rapid emergence of antibiotic resistance poses a serious public health problem worldwide. Fluoroquinolone resistance in
is an intriguing ...case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage and has remained rare. Our data indicate that deleterious fitness costs in the mammalian host constrain the emergence of fluoroquinolone resistance both by
mutation and recombination.
was able to circumvent such deleterious fitness costs via the development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, resulting in a fitness benefit during infection of mice treated with fluoroquinolones. These data suggest that the emergence of fluoroquinolone resistance is tightly constrained in
by fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes are an important contributor to the evasion of antibiotic-mediated killing.IMPORTANCEThe increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in
, whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using
models of experimental evolution, we found that
responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.
The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic ...synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.
The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a ...clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.
Display omitted
•MBS technique achieves 100- to 900-fold enrichment of S. pneumoniae from mixed samples•Known pneumococcal serotypes can be enriched from clinical saliva samples•The MBS technique shows improvement over traditional plating and isolating techniques
Use of molecular methods has improved identification of pneumococci in saliva samples; however, isolating pneumococcus, particularly from mixed/complex samples, can be challenging. We developed a magnetic bead-based separation method to enrich samples for pneumococci in order to make isolation easier and less labor intensive.
Streptococcus pneumoniae is carried asymptomatically in healthy individuals but causes disease in vulnerable populations. York et al. describe a magnetic bead-based separation technique that can enrich for S. pneumoniae from a polymicrobial clinical sample (saliva) or from a mixed-serotype experimental sample, facilitating studies aimed at improving vaccines and treatments.
Abstract
A combination of analyses were used to characterize the changes that occur in a bacterial community present in the phyllosphere of the epiphytic resurrection fern, Polypodium polypodioides, ...as the fern rehydrates from a desiccation-resistant, physiologically inactive state. Enrichment assays showed an increase in the viable count of bacteria using labile organic substrates following rainfall. Isolates obtained from enrichments were predominantly Gram-positive bacteria affiliated with various groups of the Actinobacteria and Firmicutes. In contrast, sequencing of 16S rRNA genes clones obtained from whole community DNA revealed that much of the community was dominated by other taxa, particularly the Alphaproteobacteria. Similar isolates were obtained from both dry and hydrated P. polypodioides fronds, whereas 16S rRNA gene sequencing of community DNA revealed different ribotypes on the dry and wet fern, and an overall reduction in richness following wetting. Wetting also produced changes in phyllosphere extracellular enzyme activity, with an initial burst of activity following rainfall and a subsequent burst approximately 48 h later. These findings suggest that the resurrection fern harbors a complex phyllosphere community, and that rehydration of the fern following rainfall may act as an enrichment culture stimulating certain bacterial populations and changing the overall community structure and activity.
PDF
