Biology subjects

D'Mello, A.

Publications and source records attributed to D'Mello, A..

7 recordsLinked to original sources

bioRxiv2025

Biofilm formation during pneumococcal carriage imprints naturally acquired humoral immunity

Streptococcus pneumoniae (Spn) colonization of the nasopharynx is a prerequisite for transmission and invasive disease. To investigate how repeated asymptomatic colonization shapes immunity and influences bacterial traits, we developed the Repeated Asymptomatic Murine Pneumococcal Colonization (RAMPC) model using strains belonging to serotypes: 2 (D39), 3 (WU2), and 4 (TIGR4). Sequential colonization revealed strain- and exposure-order-dependent effects on bacterial burden, with initial colonization yielding robust carriage and subsequent exposures resulting in diminished burden and rapid clearance. Humoral profiling demonstrated antigenic imprinting: the first colonizing strain largely determined IgG and IgA specificity, with minimal diversification after repeated exposures. Reactivity was strongest for biofilm-associated antigens correlating with each strains biofilm-forming capacity. Using TIGR4 mutants deficient in biofilm formation, we confirmed that in vivo aggregate formation drives humoral responses. Human sera from naturally colonized adults mirrored these findings, favoring biofilm antigens independent from capsule. Protection was demonstrated as triple-colonized mice exhibited reduced mortality and bacteremia following pneumococcal pneumonia challenge. Moreover, the initial colonizing strain influenced protection against heterologous infection, underscoring the lasting imprint of the biofilm phenotype on immunity. Finally, IgA responses in nasal-associated lymphoid tissue paralleled serum IgA patterns, validating systemic measurements as a proxy for mucosal immunity. Collectively, these results reveal that biofilm formation during colonization is a key determinant of humoral immunity and protection, providing insight into pneumococcal biology and informing strategies to design next-generation interventions. Author summaryStreptococcus pneumoniae (Spn) is a leading cause of pneumonia, meningitis, and sepsis, yet its primary lifestyle is asymptomatic colonization of the nasopharynx. Understanding how colonization shapes immunity and bacterial physiology is critical for predicting disease risk and improving future vaccines. Using a novel murine model of repeated colonization and human sera from naturally colonized adults, we show that humoral immunity is strongly biased toward antigens expressed during biofilm growth, the predominant mode of Spn in the nasopharynx, rather than planktonic forms. This response is strain-dependent and imprinted by the first colonizing strain, influencing subsequent exposures and protection against pneumonia. Importantly, biofilm formation, not capsule, drives immune recognition revealing a key link between bacterial physiology and host immunity. These findings provide fundamental insight into pneumococcal biology and the host response, suggesting that targeting biofilm-associated antigens may improve vaccine design or strategies to prevent transmission and invasive disease.

microbiology↗

bioRxiv2024

Streamlining Marker-less Allelic Replacement in Streptococcus pneumoniae Through a Single Transformation Step Strategy: easyJanus

The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic-exchange predominantly using the kanR-rpsL cassette known as "Janus". The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target genes flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our approach involves the integration of an additional gene downstream region upstream of the Janus cassette, resulting in a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. IMPORTANCEWe introduce a groundbreaking strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings, while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.

microbiology↗

bioRxiv2023

Unveiling the Immune Dynamics of Neisseria Persistent Oral Colonization: A Roadmap for Innovative Vaccine Strategies

Commensal bacteria are crucial in maintaining host physiological homeostasis, immune system development, and protection against pathogens. Despite their significance, the factors influencing persistent bacterial colonization and their impact on the host still need to be fully understood. Animal models have served as valuable tools to investigate these interactions, but most have limitations. The bacterial genus Neisseria, which includes both commensal and pathogenic species, has been studied from a pathogenicity to humans perspective, but lacks models that study immune responses in the context of long-term persistence. Neisseria musculi, a recently described natural commensal of mice, offers a unique opportunity to study long-term host-commensal interactions. In this study, for the first time we have used this model to study the transcriptional, phenotypic, and functional dynamics of immune cell signatures in the mucosal and systemic tissue of mice in response to Neisseria musculi colonization. We found key genes and pathways vital for immune homeostasis in palate tissue, validated by flow cytometry of immune cells from lung, blood and spleen. This study offers a novel avenue for advancing our understanding of host-bacteria dynamics and may provide a platform for developing efficacious interventions against mucosal persistence by pathogenic Neisseria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/572139v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1728fdaorg.highwire.dtl.DTLVardef@426a5forg.highwire.dtl.DTLVardef@55a90forg.highwire.dtl.DTLVardef@1ce3ff6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects

Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from {Delta}cas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

microbiology↗

bioRxiv2023

Influenza A virus modulation of Streptococcus pneumoniae infection using ex vivo transcriptomics in a human primary lung epithelial cell model reveals differential host glycoconjugate uptake and metabolism

BackgroundStreptococcus pneumoniae (Spn) is typically an asymptomatic colonizer of the nasopharynx but it also causes pneumonia and disseminated disease affecting various host anatomical sites. Transition from colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as result of influenza A virus coinfection. MethodsWe investigated the pneumococcal (serotype 19F, strain EF3030) and host transcriptomes with and without influenza A virus (A/California/07 2009 pH1N1) infection at this transition. This was done using primary, differentiated Human Bronchial Epithelial Cells (nHBEC) in a transwell monolayer model at an Air-Liquid Interface (ALI), with multispecies deep RNA-seq. ResultsDistinct pneumococcal gene expression profiles were observed in the presence and absence of influenza. Influenza coinfection allowed for significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways; in totality suggesting a fundamentally altered bacterial metabolic state and greater nutrient availability when coinfecting with influenza. Surprisingly, nHBEC transcriptomes were only modestly perturbed by infection with EF3030 alone in comparison to that resulting from Influenza A infection or coinfection, which had drastic alterations in thousands of genes. Influenza infected host transcriptomes suggest significant loss of ciliary function in host nHBEC cells. ConclusionsInfluenza A virus infection of nHBEC promotes pneumococcal infection. One reason for this is an altered metabolic state by the bacterium, presumably due to host components made available as result of viral infection. Influenza infection had a far greater impact on the host response than did bacterial infection alone, and this included down regulation of genes involved in expressing cilia. We conclude that influenza infection promotes a pneumococcal metabolic shift allowing for transition from colonization to disseminated disease. Author summarySecondary Streptococcus pneumoniae bacterial infections typically occur after influenza A virus respiratory infection. Such coinfections often lead to invasive pneumococcal disease. The mechanisms involved in this process are not well understood. Here, using an ex vivo human lung bronchial epithelial cell model, we investigated the biological processes of the host and pneumococcus occurring at this niche, during coinfection with multi-species transcriptomics techniques, and in vivo mouse model experimentation. We observed stark differences in global pneumococcal metabolism in different infection states, as well as viral induced epithelial cell changes in ciliary function, potentially aiding pneumococcal dissemination. Overall, this study identified broad and targeted biological processes involved in this host-pathogen interaction.

microbiology↗

bioRxiv2022

Dual species transcriptomics reveals core metabolic and immunologic processes in the interaction between primary human neutrophils and Neisseria gonorrhoeae strains

Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe and lifelong clinical sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains that are resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs or neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing (RNA-seq) was used to define Gc and PMN transcriptional profiles alone and after infection. Three strains of Gc and three human donors transcriptional responses were assessed to characterize core host and bacterial responses. Comparative analysis of Gc transcripts revealed major overlap between the Gc response to PMNs, iron, and hydrogen peroxide; specifically, the TonB system and TonB dependent transporters (TDT) were upregulated in response to PMNs. We experimentally confirmed that induction of the iron-dependent TDT TbpB is responsive to the presence of PMNs and that tonB is required for Gc survival from PMNs. Pathway analysis of PMN transcripts induced by Gc infection revealed differential expression of genes driving pathways involved in cell adhesion and migration, inflammatory responses, and inflammation resolution. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and the anti-inflammatory prostaglandin PGE2 was confirmed to be induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of three isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease in humans.

genomics↗

bioRxiv2022

In vitro phenotypic and transcriptomic variation in Neisseria musculi morphotypes correlate with colonization variability and persistence in vivo

Asymptomatic colonization of the upper respiratory tract is a common trait of the two human restricted pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. In vivo models of pathogenic neisserial infections are heterologous systems that permit short-term colonization but do not fully recapitulate infections in humans. Studying Neisseria musculi (Nmus), an oral commensal, in laboratory mice allows investigation of Neisseria-host interactions that avoids host restriction barriers. Nmus produces smooth and rough morphotypes on solid media. We compared the in vitro phenotypes, biofilm transcriptomes, in vivo colonization patterns and burdens of the two Nmus morphotypes. We observed that the two morphotypes differ in biofilm formation, pilin production, transformation frequency, and aggregation in vitro. These phenotypes strongly correlated with differential expression of a set of genes in the Nmus biofilms including those that encoded factors for bacterial attachment. In vivo, the smooth morphotype stably colonized the oral cavities of all inoculated A/J and C57BL/6J mice at higher burdens relative to the rough. Interestingly, both morphotypes colonized the oral cavities of A/Js at higher magnitudes than in C57BL/6Js. Gut colonization by the smooth morphotype was qualitatively higher than the rough. Nasal colonization in the A/Js were transient following nasal inoculations. Collectively, our results demonstrate that colonization by Nmus can be affected by various factors including Nmus morphotypes, inoculation routes, anatomical niches, and host backgrounds. The Nmus-mouse model can use variable morphotype-host combinations to study the dynamics of neisserial asymptomatic colonization and persistence in multiple extragenital niches. IMPORTANCEAnimal models for human adapted pathogenic Neisseria spp. do not fully mimic human infections and are complicated by host restriction barriers that can hinder long-term persistence. Such barriers can be avoided by studying Neisseria spp. native to the animal host used for disease models. Neisseria musculi (Nmus) isolated from wild mice colonizes the oral cavity and gut of laboratory mice for extended periods. Nmus shares host interaction factors with species pathogenic to humans and thus provides a native system to study orthologs of factors that may facilitate asymptomatic colonization and persistence in the human upper respiratory tract. We investigated the Nmus-mouse system to compare in vitro and in vivo phenotypes of two Nmus morphotypes. Our results support the hypothesis that the two morphotypes vary in different aspects of Neisseria-host interactions. Future use of the Nmus-host system will help identify molecular mechanisms required for neisserial asymptomatic colonization, dissemination, and persistence.

microbiology↗