Biological puzzles solved by using Streptococcus pneumoniae: a historical review of the pneumococcal studies that have impacted medicine and shaped molecular bacteriology.

The major human pathogen Streptococcus pneumoniae has been the subject of intensive clinical and basic scientific study for over 140 years. In multiple instances, these efforts have resulted in major breakthroughs in our understanding of basic biological principles as well as fundamental tenets of bacterial pathogenesis, immunology, vaccinology, and genetics. Discoveries made with S. pneumoniae have led to multiple major public health victories that have saved the lives of millions. Studies on S. pneumoniae continue today, where this bacterium is being used to dissect the impact of the host on disease processes, as a powerful cell biology model, and to better understand the consequence of human actions on commensal bacteria at the population level. Herein we review the major findings, i.e., puzzle pieces, made with S. pneumoniae and how, over the years, they have come together to shape our understanding of this bacterium's biology and the practice of medicine and modern molecular biology.

Quantitative mapping of mRNA 3' ends in Pseudomonas aeruginosa reveals a pervasive role for premature 3' end formation in response to azithromycin.

Pseudomonas aeruginosa produces serious chronic infections in hospitalized patients and immunocompromised individuals, including patients with cystic fibrosis. The molecular mechanisms by which P. aeruginosa responds to antibiotics and other stresses to promote persistent infections may provide new avenues for therapeutic intervention. Azithromycin (AZM), an antibiotic frequently used in cystic fibrosis treatment, is thought to improve clinical outcomes through a number of mechanisms including impaired biofilm growth and quorum sensing (QS). The mechanisms underlying the transcriptional response to AZM remain unclear. Here, we interrogated the P. aeruginosa transcriptional response to AZM using a fast, cost-effective genome-wide approach to quantitate RNA 3' ends (3pMap). We also identified hundreds of P. aeruginosa genes with high incidence of premature 3' end formation indicative of riboregulation in their transcript leaders using 3pMap. AZM treatment of planktonic and biofilm cultures alters the expression of hundreds of genes, including those involved in QS, biofilm formation, and virulence. Strikingly, most genes downregulated by AZM in biofilms had increased levels of intragenic 3' ends indicating premature transcription termination, transcriptional pausing, or accumulation of stable intermediates resulting from the action of nucleases. Reciprocally, AZM reduced premature intragenic 3' end termini in many upregulated genes. Most notably, reduced termination accompanied robust induction of obgE, a GTPase involved in persister formation in P. aeruginosa. Our results support a model in which AZM-induced changes in 3' end formation alter the expression of central regulators which in turn impairs the expression of QS, biofilm formation and stress response genes, while upregulating genes associated with persistence.

A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.

Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAlaUGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.

The Rgg1518 transcriptional regulator is a necessary facet of sugar metabolism and virulence in Streptococcus pneumoniae.

Rggs are a group of transcriptional regulators with diverse roles in metabolism and virulence. Here, we present work on the Rgg1518/SHP1518 quorum sensing system of Streptococcus pneumoniae. The activity of Rgg1518 is induced by its cognate peptide, SHP1518. In vitro analysis showed that the Rgg1518 system is active in conditions rich in galactose and mannose, key nutrients during nasopharyngeal colonization. Rgg1518 expression is highly induced in the presence of these sugars and its isogenic mutant is attenuated in growth on galactose and mannose. When compared with other Rgg systems, Rgg1518 has the largest regulon on galactose. On galactose it controls up- or downregulation of a functionally diverse set of genes involved in galactose metabolism, capsule biosynthesis, iron metabolism, protein translation, as well as other metabolic functions, acting mainly as a repressor of gene expression. Rgg1518 is a repressor of capsule biosynthesis, and binds directly to the capsule regulatory region. Comparison with other Rggs revealed inter-regulatory interactions among Rggs. Finally, the rgg1518 mutant is attenuated in colonization and virulence in a mouse model of colonization and pneumonia. We conclude that Rgg1518 is a virulence determinant that contributes to a regulatory network composed of multiple Rgg systems.

The pneumococcal social network.

Gram-positive bacteria employ an array of secreted peptides to control population-level behaviors in response to environmental cues. We review mechanistic and functional features of secreted peptides produced by the human pathogen Streptococcus pneumoniae. We discuss sequence features, mechanisms of transport, and receptors for 3 major categories of small peptides: the double-glycine peptides, the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP)-binding peptides, and the lanthionine-containing peptides. We highlight the impact of factors that contribute to carriage and pathogenesis, specifically genetic diversity, microbial competition, biofilm development, and environmental adaptation. A recent expansion in pneumococcal peptide studies reveals a complex network of interacting signaling systems where multiple peptides are integrated into the same signaling pathway, allowing multiple points of entry into the pathway and extending information content in new directions. In addition, since peptides are present in the extracellular milieu, there are opportunities for crosstalk, quorum sensing (QS), as well as intra- and interstrain and species interactions. Knowledge on the manner that population-level behaviors contribute to disease provides an avenue for the design and development of anti-infective strategies.

Flexibility and constraint: Evolutionary remodeling of the sporulation initiation pathway in Firmicutes.

The evolution of signal transduction pathways is constrained by the requirements of signal fidelity, yet flexibility is necessary to allow pathway remodeling in response to environmental challenges. A detailed understanding of how flexibility and constraint shape bacterial two component signaling systems is emerging, but how new signal transduction architectures arise remains unclear. Here, we investigate pathway remodeling using the Firmicute sporulation initiation (Spo0) pathway as a model. The present-day Spo0 pathways in Bacilli and Clostridia share common ancestry, but possess different architectures. In Clostridium acetobutylicum, sensor kinases directly phosphorylate Spo0A, the master regulator of sporulation. In Bacillus subtilis, Spo0A is activated via a four-protein phosphorelay. The current view favors an ancestral direct phosphorylation architecture, with the phosphorelay emerging in the Bacillar lineage. Our results reject this hypothesis. Our analysis of 84 broadly distributed Firmicute genomes predicts phosphorelays in numerous Clostridia, contrary to the expectation that the Spo0 phosphorelay is unique to Bacilli. Our experimental verification of a functional Spo0 phosphorelay encoded by Desulfotomaculum acetoxidans (Class Clostridia) further supports functional phosphorelays in Clostridia, which strongly suggests that the ancestral Spo0 pathway was a phosphorelay. Cross complementation assays between Bacillar and Clostridial phosphorelays demonstrate conservation of interaction specificity since their divergence over 2.7 BYA. Further, the distribution of direct phosphorylation Spo0 pathways is patchy, suggesting multiple, independent instances of remodeling from phosphorelay to direct phosphorylation. We provide evidence that these transitions are likely the result of changes in sporulation kinase specificity or acquisition of a sensor kinase with specificity for Spo0A, which is remarkably conserved in both architectures. We conclude that flexible encoding of interaction specificity, a phenotype that is only intermittently essential, and the recruitment of kinases to recognize novel environmental signals resulted in a consistent and repeated pattern of remodeling of the Spo0 pathway.

Function of BriC peptide in the pneumococcal competence and virulence portfolio.

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

Promiscuous signaling by a regulatory system unique to the pandemic PMEN1 pneumococcal lineage.

Streptococcus pneumoniae (pneumococcus) is a leading cause of death and disease in children and elderly. Genetic variability among isolates from this species is high. These differences, often the product of gene loss or gene acquisition via horizontal gene transfer, can endow strains with new molecular pathways, diverse phenotypes, and ecological advantages. PMEN1 is a widespread and multidrug-resistant pneumococcal lineage. Using comparative genomics we have determined that a regulator-peptide signal transduction system, TprA2/PhrA2, was acquired by a PMEN1 ancestor and is encoded by the vast majority of strains in this lineage. We show that TprA2 is a negative regulator of a PMEN1-specific gene encoding a lanthionine-containing peptide (lcpA). The activity of TprA2 is modulated by its cognate peptide, PhrA2. Expression of phrA2 is density-dependent and its C-terminus relieves TprA2-mediated inhibition leading to expression of lcpA. In the pneumococcal mouse model with intranasal inoculation, TprA2 had no effect on nasopharyngeal colonization but was associated with decreased lung disease via its control of lcpA levels. Furthermore, the TprA2/PhrA2 system has integrated into the pneumococcal regulatory circuitry, as PhrA2 activates TprA/PhrA, a second regulator-peptide signal transduction system widespread among pneumococci. Extracellular PhrA2 can release TprA-mediated inhibition, activating expression of TprA-repressed genes in both PMEN1 cells as well as another pneumococcal lineage. Acquisition of TprA2/PhrA2 has provided PMEN1 isolates with a mechanism to promote commensalism over dissemination and control inter-strain gene regulation.

A novel streptococcal cell-cell communication peptide promotes pneumococcal virulence and biofilm formation.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Furthermore, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

Peptide maturation molecules act as molecular gatekeepers to coordinate cell-cell communication in Streptococcus pneumoniae.

The human pathogen Streptococcus pneumoniae (Spn) encodes several cell-cell communication systems, notably multiple members of the Rgg/SHP and the Tpr/Phr families. Until now, members of these diverse communication systems were thought to work independently. Our study reveals that the ABC transporter PptAB and the transmembrane enzyme Eep act as a molecular link between Rgg/SHP and TprA/PhrA systems. We demonstrate that PptAB/Eep activates the Rgg/SHP systems and represses the TprA/PhrA system. Specifically, they regulate the respective precursor peptides (SHP and PhrA) before these leave the cell. This dual mode of action leads to temporal coordination of these systems, producing an overlap between their respective regulons during host cell infection. Thus, we have identified a single molecular mechanism that targets diverse cell-cell communication systems in Spn. Moreover, these molecular components are encoded by many gram-positive bacteria, suggesting that this mechanism may be broadly conserved.

Environmental and genetic regulation of Streptococcus pneumoniae galactose catabolic pathways.

Efficient utilization of nutrients is crucial for microbial survival and virulence. The same nutrient may be utilized by multiple catabolic pathways, indicating that the physical and chemical environments for induction as well as their functional roles may differ. Here, we study the tagatose and Leloir pathways for galactose catabolism of the human pathogen Streptococcus pneumoniae. We show that galactose utilization potentiates pneumococcal virulence, the induction of galactose catabolic pathways is influenced differentially by the concentration of galactose and temperature, and sialic acid downregulates galactose catabolism. Furthermore, the genetic regulation and in vivo induction of each pathway differ, and both galactose catabolic pathways can be turned off with a galactose analogue in a substrate-specific manner, indicating that galactose catabolic pathways can be potential drug targets.

Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates.

BACKGROUND: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. RESULTS: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. CONCLUSIONS: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.

Detection of Influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airways surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts. Importance: The impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, and a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially-complex solutions to better mimic physiologically relevant conditions.

Detection of influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.

Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery.

Bacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogen Streptococcus pneumoniae (pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population.

MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues.

Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus-infected mouse tone, and formalin-fixed paraffin-embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

Comparative genomic analyses of 17 clinical isolates of Gardnerella vaginalis provide evidence of multiple genetically isolated clades consistent with subspeciation into genovars.

Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.

Generation of genic diversity among Streptococcus pneumoniae strains via horizontal gene transfer during a chronic polyclonal pediatric infection.

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

Secondary infection with Streptococcus pneumoniae decreases influenza virus replication and is linked to severe disease.

Secondary bacterial infection is a common complication in severe influenza virus infections. During the H1N1 pandemic of 2009, increased mortality was observed among healthy young adults due to secondary bacterial pneumonia, one of the most frequent bacterial species being Streptococcus pneumoniae (Spn). Previous studies in mice and ferrets have suggested a synergistic relationship between Spn and influenza viruses. In this study, the ferret model was used to examine whether secondary Spn infection (strains BHN97 and D39) influence replication and airborne transmission of the 2009 pandemic H1N1 virus (H1N1pdm09). Secondary infection with Spn after H1N1pdm09 infection consistently resulted in a significant decrease in viral titers in the ferret nasal washes. While secondary Spn infection appeared to negatively impact influenza virus replication, animals precolonized with Spn were equally susceptible to H1N1pdm09 airborne transmission. In line with previous work, ferrets with preceding H1N1pdm09 and secondary Spn infection had increased bacterial loads and more severe clinical symptoms as compared to animals infected with H1N1pdm09 or Spn alone. Interestingly, the donor animals that displayed the most severe clinical symptoms had reduced airborne transmission of H1N1pdm09. Based on these data, we propose an asymmetrical relationship between these two pathogens, rather than a synergistic one, since secondary bacterial infection enhances Spn colonization and pathogenesis but decreases viral titers.

Competence-Associated Peptide BriC Alters Fatty Acid Biosynthesis in Streptococcus pneumoniae.

Membrane lipid homeostasis is required for bacteria to survive in a spectrum of host environments. This homeostasis is achieved by regulation of fatty acid chain length and of the ratio of unsaturated to saturated fatty acids. In the pathogen Streptococcus pneumoniae, fatty acid biosynthesis is carried out by a cluster of fatty acid biosynthesis (fab) genes (FASII locus) whose expression is controlled by the FabT repressor. Encoded immediately downstream of the FASII locus is BriC, a competence-induced, cell-cell communication peptide that promotes biofilm development as well as nasopharyngeal colonization in a murine model of pneumococcal carriage. Here, we demonstrate that briC is cotranscribed with genes of the fab gene cluster and that a reduction of briC levels, caused by decoupling its transcription from fab gene cluster, negatively affects biofilm development. BriC elevates fabT transcription, which is predicted to alter the balance of unsaturated and saturated fatty acids produced by the pathway. We find that briC inactivation results in a decreased production of unsaturated fatty acids. This affects the membrane properties by decreasing the abundance of di-unsaturated phosphatidylglycerol molecular species. We propose that the link between BriC, FabT, and phospholipid composition contributes to the ability of S. pneumoniae to alter membrane homeostasis in response to the production of a quorum-sensing peptide. IMPORTANCE Adaptation of bacteria to their host environment is a key component of colonization and pathogenesis. As an essential component of bacterial membranes, fatty acid composition contributes to host adaptation. Similarly, cell-cell communication, which enables population level responses, also contributes to host adaptation. While much is known about the pathways that control the biosynthesis of fatty acids, many questions remain regarding regulation of these pathways and consequently the factors that affect the balance between unsaturated and saturated fatty acids. We find that BriC, a cell-cell communication peptide implicated in biofilm regulation and colonization, both is influenced by a fatty acid biosynthesis pathway and affects this same pathway. This study identifies a link between cell-cell communication, fatty acid composition, and biofilms and, in doing so, suggests that these pathways are integrated into the networks that control pneumococcal colonization and host adaptation.