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.

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.

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.

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.

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.

Pneumococcal Extracellular Vesicles Modulate Host Immunity.

Extracellular vesicles (EVs) have recently garnered attention for their participation in host-microbe interactions in pneumococcal infections. However, the effect of EVs on the host immune system remain poorly understood. Our studies focus on EVs produced by Streptococcus pneumoniae (pEVs), and reveal that pEVs are internalized by macrophages, T cells, and epithelial cells. In vitro, pEVs induce NF-κB activation in a dosage-dependent manner and polarize human macrophages to an alternative (M2) phenotype. In addition, pEV pretreatment conditions macrophages to increase bacteria uptake and such macrophages may act as a reservoir for pneumococcal cells by increasing survival of the phagocytosed bacteria. When administered systemically in mice, pEVs induce cytokine release; when immobilized locally, they recruit lymphocytes and macrophages. Taken together, pEVs emerge as critical contributors to inflammatory responses and tissue damage in mammalian hosts. IMPORTANCE Over the last decade, pathogen-derived extracellular vesicles (EVs) have emerged as important players in several human diseases. Therefore, a thorough understanding of EV-mediated mechanisms could provide novel insights into vaccine/therapeutic development. A critical question in the field is: do pathogen-derived EVs help the pathogen evade the harsh environment in the host or do they help the host to mount a robust immune response against the pathogen? This study is a step towards answering this critical question for the Gram-positive pathogen, Streptococcus pneumoniae. Our study shows that while S. pneumoniae EVs (pEVs) induce inflammatory response both in vitro and in vivo, they may also condition the host macrophages to serve as a reservoir for the bacteria.

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.

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.

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.

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 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.

TprA/PhrA Quorum Sensing System Has a Major Effect on Pneumococcal Survival in Respiratory Tract and Blood, and Its Activity Is Controlled by CcpA and GlnR.

Streptococcus pneumoniae is able to cause deadly diseases by infecting different tissues, each with distinct environmental and nutritional compositions. We hypothesize that the adaptive capabilities of the microbe is an important facet of pneumococcal survival in fluctuating host environments. Quorum-sensing (QS) mechanisms are pivotal for microbial host adaptation. We previously demonstrated that the TprA/PhrA QS system is required for pneumococcal utilization of galactose and mannose, neuraminidase activity, and virulence. We also showed that the system can be modulated by using linear molecularly imprinted polymers. Due to being a drugable target, we further studied the operation of this QS system in S. pneumoniae. We found that TprA controls the expression of nine different operons on galactose and mannose. Our data revealed that TprA expression is modulated by a complex regulatory network, where the master regulators CcpA and GlnR are involved in a sugar dependent manner. Mutants in the TprA/PhrA system are highly attenuated in their survival in nasopharynx and lungs after intranasal infection, and growth in blood after intravenous infection.

Profiling microorganisms in whole saliva of children with and without dental caries.

Objectives: Dental caries is a highly prevalent infectious disease that causes tooth decay. While no single bacterial species is causative of dental caries, the role of the oral microbiome in oral health and caries is gaining interest. The purpose of this study is to compare the major species present in whole saliva samples from caries-free and caries-active children using the IBIS Universal Biosensor. Material and Methods: The abundant microbial species in ninety-five whole saliva samples from caries-free and caries-active subjects were characterized using the IBIS Universal Biosensor. Results: Twenty-four genera and sixty-five species were detected. Candida and Streptococcus were common across samples, and often the dominant genus. While we did not observe a strong association between the most abundant species and oral health, Bacteroides thetaiotaomicron and Rothia mucilaginosa were enriched in children with active caries; while, Staphylococcus epidermidis was enriched in caries-free children. Conclusions: These study trends observed suggest that microbial markers in saliva may serve as predictors of oral health and thus aid in diagnosis and treatments for prevention of caries. Consistent with competitive interactions, we also observed negative associations between Streptococcus pneumoniae and other streptococcal species, Staphylococcus aureus and S. epidermidis, Candida and Neisseria, and Saccharomyces and Streptococcus.

Gene Expression Analysis in the Pneumococcus.

Bacterial cells modify their gene expression profiles throughout different stages of growth and in response to environmental cues. Analyses of gene expression across conditions reveal both conserved and condition-specific gene responses of bacteria to adapt to these dynamic conditions. In this chapter, we present a guide to pneumococcal RNA extraction for use in the NanoString nCounter platform. The nCounter is a highly effective method to measure gene expression of bacteria not only in a planktonic mode of growth but also in the presence of host cells where the RNA of interest represents only a small portion of the total material.

Puzzling Over the Pneumococcal Pangenome.

The Gram positive bacterium Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human host, and in the nasopharynx, sinus, and middle ear it survives as a biofilm. This mode of growth is optimal for multi-strain colonization and genetic exchange. Over the last decades, the far-reaching use of antibiotics and the widespread implementation of pneumococcal multivalent conjugate vaccines have posed considerable selective pressure on pneumococci. This scenario provides an exceptional opportunity to study the evolution of the pangenome of a clinically important bacterium, and has the potential to serve as a case study for other species. The goal of this review is to highlight key findings in the studies of pneumococcal genomic diversity and plasticity.

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.

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.

Rgg-Shp regulators are important for pneumococcal colonization and invasion through their effect on mannose utilization and capsule synthesis.

Microbes communicate with each other by using quorum sensing (QS) systems and modulate their collective 'behavior' for in-host colonization and virulence, biofilm formation, and environmental adaptation. The recent increase in genome data availability reveals the presence of several putative QS sensing circuits in microbial pathogens, but many of these have not been functionally characterized yet, despite their possible utility as drug targets. To increase the repertoire of functionally characterized QS systems in bacteria, we studied Rgg144/Shp144 and Rgg939/Shp939, two putative QS systems in the important human pathogen Streptococcus pneumoniae. We find that both of these QS circuits are induced by short hydrophobic peptides (Shp) upon sensing sugars found in the respiratory tract, such as galactose and mannose. Microarray analyses using cultures grown on mannose and galactose revealed that the expression of a large number of genes is controlled by these QS systems, especially those encoding for essential physiological functions and virulence-related genes such as the capsular locus. Moreover, the array data revealed evidence for cross-talk between these systems. Finally, these Rgg systems play a key role in colonization and virulence, as deletion mutants of these QS systems are attenuated in the mouse models of colonization and pneumonia.

Gene Acquisition by a Distinct Phyletic Group within Streptococcus pneumoniae Promotes Adhesion to the Ocular Epithelium.

Streptococcus pneumoniae (pneumococcus) displays broad tissue tropism and infects multiple body sites in the human host. However, infections of the conjunctiva are limited to strains within a distinct phyletic group with multilocus sequence types ST448, ST344, ST1186, ST1270, and ST2315. In this study, we sequenced the genomes of six pneumococcal strains isolated from eye infections. The conjunctivitis isolates are grouped in a distinct phyletic group together with a subset of nasopharyngeal isolates. The keratitis (infection of the cornea) and endophthalmitis (infection of the vitreous body) isolates are grouped with the remainder of pneumococcal strains. Phenotypic characterization is consistent with morphological differences associated with the distinct phyletic group. Specifically, isolates from the distinct phyletic group form aggregates in planktonic cultures and chain-like structures in biofilms grown on abiotic surfaces. To begin to investigate the association between genotype and epidemiology, we focused on a predicted surface-exposed adhesin (SspB) encoded exclusively by this distinct phyletic group. Phylogenetic analysis of the gene encoding SspB in the context of a streptococcal species tree suggests that sspB was acquired by lateral gene transfer from Streptococcus suis. Furthermore, an sspB deletion mutant displays decreased adherence to cultured cells from the ocular epithelium compared to the isogenic wild-type and complemented strains. Together these findings suggest that acquisition of genes from outside the species has contributed to pneumococcal tissue tropism by enhancing the ability of a subset of strains to infect the ocular epithelium causing conjunctivitis. IMPORTANCE Changes in the gene content of pathogens can modify their ability to colonize and/or survive in different body sites in the human host. In this study, we investigate a gene acquisition event and its role in the pathogenesis of Streptococccus pneumoniae (pneumococcus). Our findings suggest that the gene encoding the predicted surface protein SspB has been transferred from Streptococcus suis (a distantly related streptococcal species) into a distinct set of pneumococcal strains. This group of strains distinguishes itself from the remainder of pneumococcal strains by extensive differences in genomic composition and by the ability to cause conjunctivitis. We find that the presence of sspB increases adherence of pneumococcus to the ocular epithelium. Thus, our data support the hypothesis that a subset of pneumococcal strains has gained genes from neighboring species that enhance their ability to colonize the epithelium of the eye, thus expanding into a new niche.

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.