Biology subjects

Carneiro, J.

Publications and source records attributed to Carneiro, J..

10 recordsLinked to original sources

bioRxiv2025

Regulatory Rewiring Drives Intraspecies Competition in Bacillus subtilis

Intraspecies interactions shapes microbial community structure and evolution, yet the mechanisms determining competitive outcomes among closely related strains remain unclear. The soil bacterium Bacillus subtilis is a model for microbial social interactions, where quorum-sensing systems regulate cooperation and antagonism. Here, we take a multifaceted approach to dissect the role of quorum-sensing regulation in competitive fitness. Isolate NCIB 3610 carries a signal unresponsive RapP-PhrP module that alters quorum-sensing control and promotes faster growth. Modelling and mutant analysis demonstrate that the small differences in growth rate conferred by RapP-PhrP3610 are sufficient to drive competitive exclusion. The importance of quorum sensing control is further exemplified by experimental evolution of distinct wild isolates, which revealed recurrent mutations in the sensor kinase comP, which phenocopy complete comP or comA deletions and confer a growth-linked competitive advantage. Key quorum sensing mechanisms are abandoned even in structured microbial communities, where it might be expected that communal traits are favoured. Furthermore, a phylogenomic survey of 370 B. subtilis genomes identified disruptive comP mutations in [~]16% of isolates. However, growth rate alone does not explain all interaction outcomes as even isogenic strains with equivalent doubling times differ in competitiveness. Transcriptomic profiling and validation experiments implicated a type VII secretion system toxin as an additional effector. These findings reveal that disruption of quorum-sensing pathways, whether naturally or through selection, provides a rapid route to competitive advantage, highlighting a fundamental trade-off between communal signalling and individual fitness in microbial populations. ImportanceMicrobial competition and cooperation are key in shaping the structure and evolution of microbial communities. Our study on Bacillus subtilis, a model for microbial social interactions, reveals how alterations in cell-cell communication can enhance competitive fitness. We show, through a combination of modelling, mutational analysis, and experimental evolution, that certain strains of B. subtilis gain competitive advantage by disrupting their quorum sensing pathways, which leads to faster growth and enhanced competitiveness. Such mutations are prevalent in [~]16% of analysed genomes, underscoring their widespread evolutionary benefit under competitive conditions. Our research highlights the delicate balance between individual success and community cooperation in microbes, providing insights into microbial behaviour and potential applications in modifying microbial ecosystems.

microbiology↗

bioRxiv2023

The Escherichia coli TolC efflux pump protein is immunogenic and elicits protective antibodies

Antimicrobial resistance is an increasing worldwide public health burden that threatens to make the existent antimicrobials obsolete. Among the mechanisms of antimicrobial resistance is the overexpression of efflux pumps, such as the AcrA-AcrB-TolC which extrude diverse compounds, reducing the intracellular concentration of antimicrobials. TolC is the outer membrane protein of this pump and has recently gained attention as a therapeutic target. However, little is known about the immune response generated against the TolC protein. Here we evaluated the cellular and humoral immune response against the TolC from Escherichia coli. An in silico epitope prediction of the E. coli TolC showed several residues could bind to human antibodies, and we showed that human plasma presented anti-TolC IgG and IgA antibodies. Gram-negative infected patients presented a slight increase in anti-TolC IgM amounts, compared to controls. Recombinant E. coli TolC protein stimulated macrophages in vitro to produce nitric oxide, as well as IL-6 and TNF-, assessed by Griess assay and ELISA, respectively. Immunization of mice with TolC intraperitoneally and an in vitro re-stimulation of lymph node cells led to increased percentage of T cell proliferation and IFN{gamma} production, evaluated by flow cytometry and ELISA, respectively. We observed that TolC mouse immunization stimulated anti-TolC IgM and IgG production, with a higher level of IgG1 and IgG2, amongst the IgG subclasses. Finally, TolC IgG from mouse immune serum could bind to live E. coli, increase bacterial uptake by macrophages in vitro. TolC immunized mice had a survival rate increased in 60% post infection with E. coli. Our results showed that TolC is immunogenic, activating macrophages, T and B cells, leading to the production of protective antibodies against E. coli.

immunology↗

bioRxiv2021

Discrete dynamic model of the mammalian sperm acrosome reaction: the influence of acrosomal pH and biochemical heterogeneity

The acrosome reaction (AR) is an exocytotic process essential for mammalian fertilization. It involves diverse biochemical and physiological changes that culminate in the release of the acrosomal content to the extracellular medium as well as a reorganization of the plasma membrane (PM) that allows sperm to interact and fuse with the egg. In spite of many efforts, there are still important pending questions regarding the molecular mechanism regulating the AR. Particularly, the contribution of acrosomal alkalinization to AR triggering in physiological conditions is not well understood. Also, the dependence of the proportion of sperm capable of undergoing AR on the biochemical heterogeneity within a sperm population has not been studied. Here we present a discrete mathematical model for the human sperm AR, based on the biophysical and biochemical interactions among some of the main components of this complex exocytotic process. We show that this model can qualitatively reproduce diverse experimental results, and that it can be used to analyze how acrosomal pH (pHa) and cell heterogeneity regulate AR. Our results confirm that pHa increase can on its own trigger AR in a subpopulation of sperm, and furthermore, it indicates that this is a necessary step to trigger acrosomal exocytosis through progesterone, a known physiological inducer of AR. Most importantly, we show that the proportion of sperm undergoing AR is directly related to the detailed structure of the population biochemical heterogeneity.

systems biology↗

bioRxiv2020

A first-takes-all model of centriole copy number control

How cells control the numbers of its subcellular components is a fundamental question in biology. Given that biosynthetic processes are fundamentally stochastic it is utterly puzzling that some structures display no copy number variation within a cell population. Centriole biogenesis, with each centriole being duplicated once and only once per cell cycle, stands out due to its remarkable fidelity. This is a highly controlled process, which depends on low-abundance rate-limiting factors. How can exactly one centriole copy be produced given the natural variation in the concentration of these key players? Hitherto, tentative explanations of this control evoked lateral inhibition-or phase separation-like mechanisms emerging from the dynamics of these rate-limiting factors, but how centriole number is regulated remains unclear. Here, we propose a novel solution to centriole copy number control based on the assembly of a centriolar scaffold, the cartwheel. We hypothesise that once the first cartwheel is formed it continues to elongate by stacking the intermediate cartwheel building blocks that would otherwise form supernumerary structures. Using probability theory and computer simulations, we show that this mechanism may ensure formation of one and only one cartwheel over a wide range of parameter values at physiologically relevant conditions. By comparison to alternative models, we conclude that the key signatures of this novel mechanism are an increasing assembly time with cartwheel numbers and that stochasticity in cartwheel building blocks should be converted into variation in cartwheel numbers or length, both of which can be tested experimentally. Author summaryCentriole duplication stands out as a biosynthetic process of exquisite fidelity in the noisy world of the cell. Each centriole duplicates exactly once per cell cycle, such that the number of centrioles per cell shows no variance across cells, in contrast with most cellular components that show broadly distributed copy numbers. We propose a new solution to the number control problem. We show that elongation of the first cartwheel, a core centriolar structure, may sequester the building blocks necessary to assemble supernumerary centrioles. As a corollary, the variation in regulatory kinases and cartwheel components across the cell population is predicted to translate into cartwheel length variation instead of copy number variation, which is an intriguing overlooked possibility.

cell biology↗

bioRxiv2020

Plk4 triggers autonomous de novo centriole biogenesis and maturation

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell-types, centrioles assemble de novo, yet by poorly understood mechanisms. Here, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at high Plk4 concentration, centrioles form de novo, mature and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the kinetics of centriole assembly. Moreover, our results suggest Plk4 operates in a switch-like manner to control the onset of de novo centriole formation, and that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and reveal that proteins of the pericentriolar matrix (PCM) promote biogenesis, likely by locally concentrating critical components.

cell biology↗

bioRxiv2020

CoV2ID: Detection and Therapeutics Oligo Database for SARS-CoV-2

The ability to detect the SARS-CoV-2 in a widespread epidemic is crucial for screening of carriers and for the success of quarantine efforts. Methods based on real-time reverse transcription polymerase chain reaction (RT-qPCR) and sequencing are being used for virus detection and characterization. However, RNA viruses are known for their high genetic diversity which poses a challenge for the design of efficient nucleic acid-based assays. The first SARS-CoV-2 genomic sequences already showed novel mutations, which may affect the efficiency of available screening tests leading to false-negative diagnosis or inefficient therapeutics. Here we describe the CoV2ID (http://covid.portugene.com/), a free database built to facilitate the evaluation of molecular methods for detection of SARS-CoV-2 and treatment of COVID-19. The database evaluates the available oligonucleotide sequences (PCR primers, RT-qPCR probes, etc.) considering the genetic diversity of the virus. Updated sequences alignments are used to constantly verify the theoretical efficiency of available testing methods. Detailed information on available detection protocols are also available to help laboratories implementing SARS-CoV-2 testing.

bioinformatics↗

bioRxiv2019

A web-based platform of nucleotide sequence alignments of plants

In recent years, a large number of nucleotide sequences have become available for plant species by the advent of massive parallel sequencing. The use of genomic data has been important for agriculture, food science, medicine or ecology. Despite the increasing amount of data, nucleotide sequences are usually available in public databases as isolated records with some descriptive information. Researchers interested in studying a wide range of specific plant families are forced to do multiple searches, sequence downloads, data curation and sequence alignments. In order to help researches overcoming these problems, we have built a comprehensive on-line resource of curated nucleotide sequence alignments for plant research, named PlantAligDB (available at http://plantaligdb.portugene.com). The latest release incorporates 514 alignments with a total of 66,052 sequences from six important genomic regions: atpF-atpH, psbA-trnH, trnL, rbcL, matK and ITS. The alignments represent 223 plant families from a variety of taxonomic groups. The users can quickly search the database, download and visualize the curated alignments and phylogenetic trees using dynamic browser-based applications. Different measures of genetic diversity are also available for each plant family. We also provide the workflow script that allows the user to do the curation process, explaining the steps involved. Overall, the PlantAligDB provides a complete, quality checked and regularly updated collection of alignments that can be used in taxonomic, DNA barcoding, molecular genetics, phylogenetic and evolutionary studies.

bioinformatics↗

bioRxiv2019

Partitioning stable and unstable expression level variation in cell populations: a theoretical framework with application to the T cell receptor

Phenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time,{tau} T, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance [Formula]. Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.

systems biology↗

bioRxiv2018

Interplay scenarios between CatSper and CaV channels in sea urchin sperm flagellum signaling cascades triggered by egg peptides: a mathematical modelling approach

Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of cytosolic [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelop and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation/repolarisation produced by an upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to pHi, voltage and [Ca2+]i fluctuations. The upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelop and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent, [Ca2+]i-inactivated CatSper dynamics alone. The model predicts that these two modules interfere with each other and produce unreasonable dynamics when present at similar proportions, which suggests that one may predominate over the other in vivo. To assess these alternatives, several quantitative predictions were derived from each module and confronted to experimental observations. We show that the [Ca2+]I dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper module but not by one including the pH-independent CaV and BK module. We conclude in favour of the module containing CatSper.

developmental biology↗

bioRxiv2017

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

biophysics↗