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Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions

Rueff, Anne-Stéphanie; van Raaphorst, Renske; Aggarwal, Surya D; Santos-Moreno, Javier; Laloux, Géraldine; Schaerli, Yolanda; Weiser, Jeffrey N; Veening, Jan-Willem
Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.
PMID: 37978173
ISSN: 2041-1723
CID: 5610712

Effects of Capsular Polysaccharide amount on Pneumococcal-Host interactions

Zhu, Jiaqi; Abruzzo, Annie R; Wu, Cindy; Bee, Gavyn Chern Wei; Pironti, Alejandro; Putzel, Gregory; Aggarwal, Surya D; Eichner, Hannes; Weiser, Jeffrey N
Among the many oral streptococci, Streptococcus pneumoniae (Spn) stands out for the capacity of encapsulated strains to cause invasive infection. Spread beyond upper airways, however, is a biological dead end for the organism, raising the question of the benefits of expending energy to coat its surface in a thick layer of capsular polysaccharide (CPS). In this study, we compare mutants of two serotypes expressing different amounts of CPS and test these in murine models of colonization, invasion infection and transmission. Our analysis of the effect of CPS amount shows that Spn expresses a capsule of sufficient thickness to shield its surface from the deposition of complement and binding of antibody to underlying epitopes. While effective shielding is permissive for invasive infection, its primary contribution to the organism appears to be in the dynamics of colonization. A thicker capsule increases bacterial retention in the nasopharynx, the first event in colonization, and also impedes IL-17-dependent clearance during late colonization. Enhanced colonization is associated with increased opportunity for host-to-host transmission. Additionally, we document substantial differences in CPS amount among clinical isolates of three common serotypes. Together, our findings show that CPS amount is highly variable among Spn and could be an independent determinant affecting host interactions.
PMID: 37540710
ISSN: 1553-7374
CID: 5595372

Rewilding of laboratory mice enhances granulopoiesis and immunity through intestinal fungal colonization

Chen, Ying-Han; Yeung, Frank; Lacey, Keenan A; Zaldana, Kimberly; Lin, Jian-Da; Bee, Gavyn Chern Wei; McCauley, Caroline; Barre, Ramya S; Liang, Shen-Huan; Hansen, Christina B; Downie, Alexander E; Tio, Kyle; Weiser, Jeffrey N; Torres, Victor J; Bennett, Richard J; Loke, P'ng; Graham, Andrea L; Cadwell, Ken
The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component β-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.
PMID: 37352372
ISSN: 2470-9468
CID: 5537252

Age-dependent differences in efferocytosis determine the outcome of opsonophagocytic protection from invasive pathogens

Bee, Gavyn Chern Wei; Lokken-Toyli, Kristen L; Yeung, Stephen T; Rodriguez, Lucie; Zangari, Tonia; Anderson, Exene E; Ghosh, Sourav; Rothlin, Carla V; Brodin, Petter; Khanna, Kamal M; Weiser, Jeffrey N
In early life, susceptibility to invasive infection skews toward a small subset of microbes, whereas other pathogens associated with diseases later in life, including Streptococcus pneumoniae (Spn), are uncommon among neonates. To delineate mechanisms behind age-dependent susceptibility, we compared age-specific mouse models of invasive Spn infection. We show enhanced CD11b-dependent opsonophagocytosis by neonatal neutrophils improved protection against Spn during early life. The augmented function of neonatal neutrophils was mediated by higher CD11b surface expression at the population level due to dampened efferocytosis, which also resulted in more CD11bhi "aged" neutrophils in peripheral blood. Dampened efferocytosis during early life could be attributed to the lack of CD169+ macrophages in neonates and reduced systemic expressions of multiple efferocytic mediators, including MerTK. On experimentally impairing efferocytosis later in life, CD11bhi neutrophils increased and protection against Spn improved. Our findings reveal how age-dependent differences in efferocytosis determine infection outcome through the modulation of CD11b-driven opsonophagocytosis and immunity.
PMID: 37059107
ISSN: 1097-4180
CID: 5502802

BlpC-mediated selfish program leads to rapid loss of Streptococcus pneumoniae clonal diversity during infection

Aggarwal, Surya D; Lees, John A; Jacobs, Nathan T; Bee, Gavyn Chern Wei; Abruzzo, Annie R; Weiser, Jeffrey N
Successful colonization of a host requires bacterial adaptation through genetic and population changes that are incompletely defined. Using chromosomal barcoding and high-throughput sequencing, we investigate the population dynamics of Streptococcus pneumoniae during infant mouse colonization. Within 1 day post inoculation, diversity was reduced >35-fold with expansion of a single clonal lineage. This loss of diversity was not due to immune factors, microbiota, or exclusive genetic drift. Rather, bacteriocins induced by the BlpC-quorum sensing pheromone resulted in predation of kin cells. In this intra-strain competition, the subpopulation reaching a quorum likely eliminates others that have yet to activate the blp locus. Additionally, this reduced diversity restricts the number of unique clones that establish colonization during transmission between hosts. Genetic variation in the blp locus was also associated with altered transmissibility in a human population, further underscoring the importance of BlpC in clonal selection and its role as a selfish element.
PMID: 36395758
ISSN: 1934-6069
CID: 5384942

Oxidative Reactions Catalyzed by Hydrogen Peroxide Produced by Streptococcus pneumoniae and Other Streptococci Cause the Release and Degradation of Heme from Hemoglobin

Alibayov, Babek; Scasny, Anna; Khan, Faidad; Creel, Aidan; Smith, Perriann; Vidal, Ana G Jop; Fitisemanu, Fa'alataitaua M; Padilla-Benavides, Teresita; Weiser, Jeffrey N; Vidal, Jorge E
Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin, but oxidative reactions catalyzed by Spn-H2O2 have been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single-mutant (ΔspxB or ΔlctO), double-mutant (ΔspxB ΔlctO), and complemented strains in TIGR4, D39, and EF3030. We then utilized an in vitro model with oxyhemoglobin to demonstrate that oxyhemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to methemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacterial inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation, whereas bacterial species that produce <1 μM H2O2 neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin, induced upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.
PMID: 36409115
ISSN: 1098-5522
CID: 5384062

Serotype-Dependent Effects on the Dynamics of Pneumococcal Colonization and Implications for Transmission

Abruzzo, Annie R; Aggarwal, Surya D; Sharp, Molly E; Bee, Gavyn Chern Wei; Weiser, Jeffrey N
Capsule-switch mutants were compared to analyze how serotype affects the success of Streptococcus pneumoniae (Spn) during colonization and transmission. Strains of multiple serotypes were tested in highly susceptible infant mice, both singly and in competitive assays. Our findings demonstrated a role of serotype, apart from genetic background, in competitive success of strains, but this depended on timing postinoculation. As is the case for natural carriage, there was a hierarchy of success among serotypes using capsule-switch strains. The long-term dominance of a serotype was established within the first 4 h after acquisition, suggesting an effect independent of Spn-induced host responses. The hierarchy of serotype dominance correlated with decreased clearance rather than increased growth in vivo. Competitive assays staggering the timing of challenge showed that the first strain to dominate the niche sustained its competitive advantage, potentially explaining how increased density from delayed early clearance could result in serotype-dependent success. Effector molecules of intrastrain competition (fratricide), regulated by the competence operon in a quorum-sensing mechanism, were required for early niche dominance. This suggested a winner-takes-all scenario in which serotype is a major factor in achieving early niche dominance, such that once a strain reaches a threshold density it is able to exclude competitors through fratricide. Serotype was also an important determinant of transmission dynamics, although transit to a recipient host depended on effects of serotype different from its contribution to the dominance of colonization in the donor host. IMPORTANCE Capsule is the major virulence factor and surface antigen of the opportunistic respiratory pathogen Streptococcus pneumoniae (Spn). Strains of Spn express at least 100 structurally and immunologically distinct types (serotypes) of capsule, but for unknown reasons only a few are common. The effect of serotypes during the commensal interactions of Spn and its host, colonization and transmission, was tested. This was carried out by comparing genetically modified strains differing only in serotype in infant mouse models. Results show that serotype is an important factor in a strain's success during colonization. This was attributed to the effect of serotype on early clearance of the organism in the host. Competitive factors expressed by Spn (in a mechanism referred to as fratricide) allow the strain gaining this initial advantage to then dominate the upper respiratory tract niche. Serotype also plays an important role in a strain's success during transmission from one host to another.
PMID: 35289642
ISSN: 2150-7511
CID: 5216812

Pneumococcal capsule blocks protection by immunization with conserved surface proteins

Zangari, Tonia; Zafar, M Ammar; Lees, John A; Abruzzo, Annie R; Bee, Gavyn Chern Wei; Weiser, Jeffrey N
Vaccines targeting Streptococcus pneumoniae (Spn) are limited by dependence on capsular polysaccharide and its serotype diversity. More broadly-based approaches using common protein antigens have not resulted in a licensed vaccine. Herein, we used an unbiased, genome-wide approach to find novel vaccine antigens to disrupt carriage modeled in mice. A Tn-Seq screen identified 198 genes required for colonization of which 16 are known to express conserved, immunogenic surface proteins. After testing defined mutants for impaired colonization of infant and adult mice, 5 validated candidates (StkP, PenA/Pbp2a, PgdA, HtrA, and LytD/Pce/CbpE) were used as immunogens. Despite induction of antibody recognizing the Spn cell surface, there was no protection against Spn colonization. There was, however, protection against an unencapsulated Spn mutant. This result correlated with increased antibody binding to the bacterial surface in the absence of capsule. Our findings demonstrate how the pneumococcal capsule interferes with mucosal protection by antibody to common protein targets.
PMID: 34930916
ISSN: 2059-0105
CID: 5108742

Decreased production of epithelial-derived antimicrobial molecules at mucosal barriers during early life

Lokken-Toyli, Kristen L; de Steenhuijsen Piters, Wouter A A; Zangari, Tonia; Martel, Rachel; Kuipers, Kirsten; Shopsin, Bo; Loomis, Cynthia; Bogaert, Debby; Weiser, Jeffrey N
Young age is a risk factor for respiratory and gastrointestinal infections. Here, we compared infant and adult mice to identify age-dependent mechanisms that drive susceptibility to mucosal infections during early life. Transcriptional profiling of the upper respiratory tract (URT) epithelium revealed significant dampening of early life innate mucosal defenses. Epithelial-mediated production of the most abundant antimicrobial molecules, lysozyme, and lactoferrin, and the polymeric immunoglobulin receptor (pIgR), responsible for IgA transcytosis, was expressed in an age-dependent manner. This was attributed to delayed functional development of serous cells. Absence of epithelial-derived lysozyme and the pIgR was also observed in the small intestine during early life. Infection of infant mice with lysozyme-susceptible strains of Streptococcus pneumoniae or Staphylococcus aureus in the URT or gastrointestinal tract, respectively, demonstrated an age-dependent regulation of lysozyme enzymatic activity. Lysozyme derived from maternal milk partially compensated for the reduction in URT lysozyme activity of infant mice. Similar to our observations in mice, expression of lysozyme and the pIgR in nasopharyngeal samples collected from healthy human infants during the first year of life followed an age-dependent regulation. Thus, a global pattern of reduced antimicrobial and IgA-mediated defenses may contribute to increased susceptibility of young children to mucosal infections.
PMID: 34465896
ISSN: 1935-3456
CID: 4998412

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary Th17 Response that Mitigates Susceptibility to Streptococcus pneumoniae

Wu, Benjamin G; Sulaiman, Imran; Tsay, Jun-Chieh J; Perez, Luisanny; Franca, Brendan; Li, Yonghua; Wang, Jing; Gonzalez, Amber N; El-Ashmawy, Mariam; Carpenito, Joseph; Olsen, Evan; Sauthoff, Maya; Yie, Kevin; Liu, Xiuxiu; Shen, Nan; Clemente, Jose C; Kapoor, Bianca; Zangari, Tonia; Mezzano, Valeria; Loomis, Cynthia; Weiden, Michael D; Koralov, Sergei; D'Armiento, Jeanine; Ahuja, Sunil K; Wu, Xue-Ru; Weiser, Jeffrey N; Segal, Leopoldo N
Rationale Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with increased Th17 inflammatory phenotype. In this study we evaluated the microbial and host immune response dynamics after aspiration with a oral commensals using a preclinical mouse model. Methods Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of sacrifice. Genetic background of mice included WT, MyD88 knock out and STAT3C. Measurements 16S rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host transcriptome sequencing was used to characterize host immune phenotype. Main Results While MOC aspiration correlated with lower airway dysbiosis that resolved within five days, it induced an extended inflammatory response associated with IL17-producing T-cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration prior to a respiratory challenge with S. pneumoniae led to a decreased in host's susceptibility to this pathogen. Conclusions Thus, in otherwise healthy mice, a single aspiration event with oral commensals are rapidly cleared from the lower airways, but induce a prolonged Th17 response that secondarily decreased susceptibility to respiratory pathogens. Translationally, these data implicate an immuno-protective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower airway pathogens.
PMID: 33166473
ISSN: 1535-4970
CID: 4664852