Biology subjects

Aggarwal, S. D.

Publications and source records attributed to Aggarwal, S. D..

6 recordsLinked to original sources

bioRxiv2023

Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions

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 virulence1-8, 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 outcomes9-14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15-20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy 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.

microbiology↗

bioRxiv2022

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

Chromosomal barcoding and high-throughput sequencing were used to investigate the population dynamics of Streptococcus pneumoniae. During infant mouse colonization, >35-fold reduction in diversity and expansion of a single clonal lineage was observed within 1 day post-inoculation. This loss of diversity was not due to immune factors, host microbiota or exclusively because of genetic drift. Rather, it required the expression of blp bacteriocins induced by the BlpC-quorum sensing pheromone. This points towards the role of intra-strain competition whereby the subpopulation reaching a quorum eliminates others that have yet to activate the blp locus. We show that this loss of diversity also restricts the number of unique clones that could establish colonization during transmission between hosts. Moreover, we show that genetic variation in the blp locus is associated with transmissibility in the human population. We posit this is due to its importance in clonal selection and its role as a selfish element.

microbiology↗

bioRxiv2021

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 saturated to unsaturated fatty acids. In the pathogen Streptococcus pneumoniae, fatty acid biosynthesis is encoded 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 co-transcribed with genes of the fab gene cluster and that a reduction of briC levels, caused by decoupling its transcription from fab gene cluster, negatively impacts biofilm development. BriC elevates fabT transcription, which is predicted to alter the balance of saturated and unsaturated fatty acids produced by the pathway. We find that briC inactivation results in a decreased production of unsaturated fatty acids that impact 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. IMPORTANCEAdaptation 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, so does cell-cell communication, which serves as a mechanism for population levels responses. 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 impacts the balance between saturated and unsaturated fatty acids. We find that BriC, a cell-cell communication peptide implicated in biofilm regulation and colonization, is both influenced by a fatty acid biosynthesis pathway and impacts this same pathway. This study identified 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.

microbiology↗

bioRxiv2020

Neuraminidase B controls neuraminidase A-dependent mucus production and evasion

Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions. Author SummaryStreptococcus pneumoniae (Spn) is a leading mucosal pathogen, whose host interaction begins with colonization of the upper respiratory tract. While there has been extensive investigation into bacterial interaction with epithelial cells, there is little understanding of bacterial-mucus interactions. Our study used mucus of human and murine origin and a murine model of colonization to study mucus associations involving Spn. The main findings reveal i) the enzymatic activity of Spns neuraminidases (NanA and NanB) contribute to mucus evasion through removing terminal sialic acid, ii) the enzymatic activity of NanB controls expression of the main neuraminidase, NanA, and iii) Spn induces sialic acid containing mucus secretions in vivo in a neuraminidase-dependent manner. We postulate that during colonization, neuraminidase-dependent reduction in mucus binding enables evasion of mucociliary clearance, which is necessary to counter neuraminidase-mediated stimulation of mucus secretions. Thus, our study provides new insights into the role of Spn neuraminidases on colonization.

microbiology↗

bioRxiv2019

A Molecular Link between Cell Wall Modification and Stringent Response in a Gram-positive Bacteria

To ensure survival during colonization of the human host, bacteria must successfully respond to unfavorable and fluctuating conditions. This study explores the fundamental phenomenon of stress response in a gram-positive bacterium, where we investigate the ability of a cell wall modification enzyme to modulate intracellular stress and prevent the triggering of the stringent response pathway. The Streptococcus pneumoniae cell wall modification proteins MurM and MurN are tRNA-dependent amino acid ligases, which lead to the production of branched muropeptides by generating peptide crossbridges. In addition, MurM has been proposed to contribute to translation quality control by preferentially deacylating mischarged tRNAs mischarged with amino acids that make up the peptidoglycan. Here, we demonstrate that the murMN operon promotes optimal growth under stressed conditions. Specifically, when grown in mildly acidic conditions, a murMN deletion mutant displays early entry into stationary phase and dramatically increased lysis. Surprisingly, these defects are rescued by inhibition of the stringent response pathway or by enhancement of the cells ability to deacylate mischarged tRNA molecules. The increase in lysis results from the activity of LytA, and experiments in macrophages reveal that murMN regulates phagocytosis in a LytA-dependent manner. These results suggest that under certain stresses, these bacterial cells lacking MurMN likely accumulate mischarged tRNA molecules, activate the stringent response pathway, and enter prematurely into stationary phase. Moreover, by virtue of its ability to deacylate mischarged tRNAs while building peptidoglycan crossbridges, MurM can calibrate the stress response with consequences to host-pathogen interactions. Thus, MurM is positioned at the interface of cell wall modification, translation quality control and stringent response. These findings expand our understanding of the functions of the bacterial cell wall: cell wall modifications that impart structural rigidity to the cell are interlinked to the cells ability to signal intracellularly and mount a response to environmental stresses.\n\nSIGNIFICANCEDuring infection, microbes must survive the hostile environmental conditions of the human host. When exposed to stresses, bacteria activate an intracellular response, known as stringent response pathway, to ensure their survival. This study connects two fundamental pathways important for cellular growth in a gram-positive bacterium; it demonstrates that enzymes responsible for cell wall modification are connected to the stringent response pathway via their ability to ameliorate errors in protein translation. Our study was performed on Streptococcus pneumoniae where the cell wall modification enzyme, MurM, is a known determinant of penicillin resistance. We now demonstrate the importance of MurM in translation quality control and establish that it serves as a gatekeeper of the stringent response pathway.

microbiology↗

bioRxiv2018

The Streptococcus pneumoniae Competence-Induced BriC Peptide Promotes Nasopharyngeal Colonization and Impacts Biofilm Development

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.

microbiology↗