Biology subjects

Henriques, A. O.

Publications and source records attributed to Henriques, A. O..

10 recordsLinked to original sources

bioRxiv2026

Hierarchical cross-linking of a bacterial spore coat Hub protein

Hub proteins are highly connected nodes in protein-protein interaction networks and are often intrinsically disordered proteins (IDPs) or contain intrinsically disordered regions. In Bacillus subtilis, the morphogenesis of the spore surface is orchestrated by a set of so-called morphogenetic proteins that guide the assembly of distinct layers. Formation of the inner coat is directed by SafAFL and its shorter isoform, C30. Both are expressed early in sporulation under the control of {sigma}E and localize at the interface between the developing inner coat and the underlying cortex peptidoglycan. From this site, they act as organizational hubs, recruiting client proteins essential for coat maturation. Among these is Tgl, a transglutaminase synthesized later in development following activation of {sigma}K after engulfment completion. We show that the C30 domain exhibits IDP-like features yet self-assembles into >1200 kDa complexes stabilized by disulfide bonds and that these bonds are required for subsequent proper Tgl-mediated "spotwelding" cross-linking. Small-angle X-ray scattering (SAXS) and photobleaching show that Tgl immobilizes but does not drastically alter these assemblies. These findings support a hierarchical, biphasic model for inner coat assembly: initial self-assembly and disulfide stabilization, followed by Tgl-mediated cross-linking and structural stabilization. According to this model, the forms of SafAFL/C30 that dominate the two stages recruit different client proteins in register with the course of morphogenesis.

microbiology↗

bioRxiv2025

Streptolancidin D mediates intra-species competition in Streptococcus pneumoniae through the Rgg144/SHP144 quorum-sensing system

Streptococcus pneumoniae is a major colonizer of the human nasopharynx, where inter- and intra-strain competition plays a critical role in shaping population structure and influencing vaccine outcomes. Bacteriocins are key mediators of intra-species competition, yet many of their functions and regulatory mechanisms remain poorly understood. Here, we identify and characterize streptolancidin D, a previously uncharacterized bacteriocin encoded by the sldA-T locus and demonstrate its contribution to pneumococcal competition. Using isogenic streptolancidin-producing and non-producing variants of a naturally colonizing strain, we show that sldA-T contributes to inhibition of competitor strains in in vitro biofilms and during murine co-colonization. Importantly, streptolancidin D also inhibited a subset of genetically diverse pneumococcal isolates representing multiple serotypes, whereas non-producing variants showed no activity. This indicates that its effect is broad and not restricted to isogenic interactions. Genomic analysis of over 7,500 pneumococcal genomes revealed that sldA-T is present in [~]12% of isolates, with lineage-associated distribution patterns, and is consistently encoded downstream of the Rgg144/SHP144 quorum sensing system. We further demonstrate that sldA-T is regulated by this system, with sldA-T promoter activity abolished in a SHP-deficient background and partially restored by exogenous peptide stimulation. Finally, we show that streptolancidin D acts in concert with other bacteriocin systems and competence-mediated fratricide, highlighting a multifactorial antimicrobial strategy that enhances pneumococcal competitiveness. Overall, our findings identify a quorum sensing-regulated bacteriocin that contributes to pneumococcal competition and helps shape population dynamics. IMPORTANCEBacteriocins are central to bacterial competition and niche occupation, particularly in structured environments like the human nasopharynx. While several pneumococcal bacteriocins have been characterized, the functions of many remain unknown, limiting our understanding of how these systems shape strain fitness and population dynamics. We characterize streptolancidin D, a bacteriocin that enhances intraspecies competitiveness in vitro and in vivo and contributes to inhibition of genetically diverse pneumococcal strains. We demonstrate that its expression is tightly regulated by the conserved Rgg144/SHP144 quorum sensing system and that the locus is distributed and shows synteny across multiple pneumococcal lineages. Our findings reveal that streptolancidin D operates within a broader network of bacteriocins and competence-associated mechanisms that collectively shape competitive interactions. By integrating genomic, functional, and regulatory analyses, this work expands the known repertoire of pneumococcal antimicrobial systems and provides new insights into the mechanisms underpinning competition and population structure in S. pneumoniae.

microbiology↗

bioRxiv2024

The impact of YabG mutations on C. difficile spore germination and processing of spore substrates

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (catalytically inactive), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

microbiology↗

bioRxiv2023

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

The pathogenic bacteria Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the {sigma}K-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of {sigma}K is not strictly linked to the state of forespore development in B. cereus.

microbiology↗

bioRxiv2020

The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates interactions of spores with their environment, modulates spore adhesion and germination and could be implicated in pathogenesis. The exosporium is composed of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, and surrounded by a glycoprotein hairy nap. The morphogenetic protein CotE is necessary for Bacillus cereus exosporium integrity, but how CotE directs exosporium assembly remains unknown. Here, we followed the localization of SNAP-tagged CotE, -CotY and -ExsY during B. cereus sporulation, using super-resolution fluorescence microscopy and evidenced interactions among these proteins. CotE, CotY and ExsY are present as complexes at all sporulation stages and follow a similar localization pattern during endospore formation that is reminiscent of the localization of Bacillus subtilis CotE. We show that B. cereus CotE drives the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process as previously proposed but it is performed through the formation of two caps, as observed during B. subtilis coat morphogenesis. It appears that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCESpores of Bacillaceae are enveloped in a glycoprotein outermost layer. In the B. cereus group, encompassing the B. anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance separated from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize formation of the exosporium during sporulation of B. cereus and we studied the localization and interactions of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaf-fold, before a complete exosporium or coat are detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore and we present a model for the assembly of the exosporium.

microbiology↗

bioRxiv2020

A regulatory protein that represses sporulation in Clostridioides difficile

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50 to 60% of those bacteria have the ability to produce resilient spores, important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile, and contains several genes of unknown function. We report on the characterization of a signature gene, csiA, which, as we show, is involved in the control of sporulation initiation in C. difficile. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of csiA results in increased sporulation, and increased expression of spo0A per cell. Spo0A also drives transcription of the spoIIA and spoIIG operons, coding for the first forespore-({sigma}F) and mother cell-specific ({sigma}E) RNA polymerase sigma factors. Strikingly, deletion of csiA increases expression of the spoIIG operon, but not that of the spoIIA operon. Increased expression of spoIIG results in increased production and proteolytic activation of pro-{sigma}E, suggesting that normally, the levels of active {sigma}E are limiting for sporulation. While other regulatory proteins affect both sporulation and several processes during the transition phase of growth, including toxin production or motility, deletion of the csiA gene does not alter the expression of the genes coding for the TcdA and TcdB cytotoxins or the genes involved in motility. Thus, our results establish that CsiA acts to modulate sporulation by reducing expression of the spo0A gene.

microbiology↗

bioRxiv2019

Rampant loss of social traits during domestication of a wild Bacillus subtilis

Most well-studied bacteria have been domesticated to some extent. How fast can a natural isolate diverge from its ancestral phenotypes under domestication to a novel laboratory environment is poorly known. Yet such information is key to understand rates of evolution, the time scale at which a natural isolate can be propagated without loss of its natural adaptive traits and the reliability of experimental results across labs. Using experimental evolution, phenotypic assays and whole-genome sequencing, we show that within a week of propagation in a common laboratory environment, a natural isolate of Bacillus subtilis acquires mutations that cause changes in a multitude of traits. A single adaptive mutational step, in the gene coding for the transcriptional regulator DegU, impairs a DegU-dependent positive autoregulatory loop and leads to loss of robust biofilm architecture, impaired swarming motility, reduced secretion of exoproteases and changes in the dynamics of sporulation across environments. Importantly, domestication also resulted in improved survival when the bacteria face pressure from cells of the innate immune system. These results show that degU is a key target for mutations during domestication and also underscore the importance of performing careful and extremely short-term propagations of natural isolates to conserve the traits encoded in their original genomes. SummaryDomestication is the process by which organisms are selected to live in specific conditions and an important phenomenon that shapes the evolution and variation in many animals and plants. In microbes, domestication is also a key driver of adaptation. It can be beneficial, when improving microbes abilities that are important for biotechnology, but also problematic, especially when studying microbe-host interactions and the microbes natural behavior. Using a natural isolate of Bacillus subtilis, we determined the speed and genetic basis of microbial domestication using experimental evolution. Within one week of growth in the common laboratory media, mutations in the pleiotropic transcriptional regulator, DegU, emerge and spread in the populations. These lead to loss of social traits, increased resistance to bacteriophages and increased survival in the presence of macrophages. The data highlights the extreme caution that is needed when culturing natural microbial isolates and may help explain why some key microbial social traits and behaviors may differ between different laboratories, even when studying the same strains.

evolutionary biology↗

bioRxiv2019

From root to tips: sporulation evolution and specialization in Bacillus subtilis and the intestinal pathogen Clostridioides difficile

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of a highly resistant, dormant spore. Spores allow environmental persistence, dissemination and for pathogens, are infection vehicles. In both the model Bacillus subtilis, an aerobic species, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation. Using the genes involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macro-evolutionary framework we show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been co-evolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli-lineage restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota and an adjustment of sporulation to this particular ecosystem.

evolutionary biology↗

bioRxiv2018

Structure and assembly of a Clostridioides difficile spore polar appendage

Clostridioides difficile, a strict anaerobic spore-former, is the main cause of nosocomial disease associated to antibiotic therapy in adults and a growing concern in the community. Spores are the main infectious, persistence and transmission vehicle. Spore germination occurs in the intestine and the resulting vegetative cells will produce the toxins responsible for the disease symptoms, and spores. During sporulation, a wild type population bifurcates into two main spore morphotypes, with or without a thick exosporium. We show that this bifurcation extends to the formation of spores with a robust polar appendage or spores with a short appendage or that lack this structure. The cysteine-rich CdeM protein localizes to the appendage and around the entire surface of the spore, and is a major structural component of the exosporium, which we show is continuous with the appendage. In a CdeM mutant, when present, the polar appendage is short and disorganized. We show that wild type and cdeM spores with a short or no appendage germinate poorly in response to taurocholate, compared to those with an appendage. cdeM spores of the two types, however, germinate faster than their wild type counterparts. Thus, while the absence of CdeM may increase the permeability of spores to taurocholate, proper assembly of the appendage is also important for germination. Consistent with an overall enhancement of germination, a cdeM mutant shows increased virulence in a hamster model of disease. For a wild type population, spores with a short or no appendage germinate slower than the appendage-bearing spores. Differences in transmission, persistence and disease severity may result, in part, from their proportion in a spore population.

microbiology↗

bioRxiv2018

A protein phosphorylation module patterns the Bacillus subtilis spore outer coat

Assembly of the Bacillus subtilis spore coat involves over 80 protein components, which self-organize into a basal layer, a lamellar inner coat, a striated electrondense outer coat and a more external crust. CotB is an abundant component of the outer coat. Its C-terminal moiety contains a region, termed SKRB, formed by a series of serine-rich repeats, which we show is phosphorylated by the coat-associated Ser/Thr kinase CotH at multiple Ser residues. Another coat protein, CotG, which contains a central repeat region, SKRG, interacts with the C-terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a cotHD288A strain, producing an inactive form of the kinase, like those formed by a cotG deletion mutant, lack the characteristic pattern of electrondense outer coat striations, while retaining the crust. Specifically, in the absence of CotG or CotH activity, most of the outer coat proteins are assembled but form a layer of amorphous material that peels-off the spore if crust formation is genetically ablated. In contrast, deletion of the SKRB region, has no major impact on the structure of the outer coat. Thus, phosphorylation of CotG by CotH is the principal factor establishing the structural pattern of the spore outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis and of cotG-like proteins in nearly all spore-formers that also code for a CotH homologue hints at the importance of this protein phosphorylation module in the morphogenesis of the spore outer layers.

microbiology↗