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Abstract Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes.

Abstract Group B Streptococcus (GBS) remains the leading bacterial cause of invasive neonatal disease, resulting in substantial morbidity and mortality. New therapeutic approaches beyond antibacterial treatment to prevent neonatal disease outcomes are urgent. One significant limitation in studying GBS disease and progression is the lack of non-invasive technologies for longitudinal studies. Here, we develop and compare three bioluminescent GBS strains for in vivo pathogenic analysis. Bioluminescence is based on the luxABCDE operon on a replicative vector (luxGBS-CC17), and the red-shifted firefly luciferase on a replicative vector (fflucGBS-CC17) or integrated in the genome (glucGBS-CC17). We show that luxGBS-CC17 is suitable for in vitro analysis but does not produce a significant bioluminescent signal in infected pups. In contrast, the fflucGBS-CC17 results in a strong bioluminescent signal proportional to the organ colonisation level. However, the stability of the replicative vector depends on the route of infection, especially when pups acquire the bacteria from infected vaginal mucosa. Stable chromosomal integration of luciferase in glucGBS-CC17 leads to significant bioluminescence in both haematological and vertical infection models associated with high systemic colonisation. These strains will allow the preclinical evaluation of treatment efficacy against GBS invasive disease using whole-mouse bioluminescence imaging.

ABSTRACT Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection by hindering the ability of bacteria to persist at the inoculation site and to spread to distant organs. Transcriptome and in vivo analysis reveal a specialized regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases PbsP-dependent adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a dynamic, highly specialized regulatory system enabling GBS to express a restricted set of virulence factors that promote invasion of host barriers and allow these bacteria to persist inside the host during lethal infection.IMPORTANCEGroup B Streptococcus (or GBS) is a normal inhabitant of the human gastrointestinal and genital tracts that can also cause deadly infections in newborns and elderly people. The transition from a harmless commensal to a dangerous pathogen relies on the timely expression of bacterial molecules necessary for causing disease. In this study, we characterize the two-component system SaeRS as a key regulator of such virulence factors. Our analysis reveals a specialized regulatory system that is activated only during infection to dynamically adjust the production of two virulence factors involved in interactions with host cells. Overall, our findings highlight the critical role of SaeRS in GBS infections and suggest that targeting this system may be useful for developing new antibacterial drugs.

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases.

Sweetpotato is an important food crop globally, serving as a rich source of carbohydrates, vitamins, fiber, and micronutrients. Sweetpotato yield depends on the modification of adventitious roots into storage roots. The underlying mechanism of this developmental switch is not fully understood. Interestingly, storage-root formation is manifested by formation of starch-accumulating parenchyma cells and bulking of the distal part of the root, while the proximal part does not show bulking. This system, where two parts of the same adventitious root display different developmental fates, was used by us in order to better characterize the anatomical, physiological, and molecular mechanisms involved in sweetpotato storage-root formation. We show that, as early as 1 and 2 weeks after planting, the proximal part of the root exhibited enhanced xylem development together with increased/massive lignin deposition, while, at the same time, the distal root part exhibited significantly elevated starch accumulation. In accordance with these developmental differences, the proximal root part exhibited up-regulated transcript levels of sweetpotato orthologs of Arabidopsis vascular-development regulators and key genes of lignin biosynthesis, while the distal part showed up-regulation of genes encoding enzymes of starch biosynthesis. All these recorded differences between proximal and distal root parts were further enhanced at 5 weeks after planting, when storage roots were formed at the distal part. Our results point to down-regulation of fiber formation and lignification, together with up-regulation of starch biosynthesis, as the main events underlying storage-root formation, marking/highlighting several genes as potential regulators, providing a valuable database of genes for further research.

A Közgazdaságtudományi Kar 20 éves fennállásának alkalmából rendezett jubileumi konferencián a délelőtt során egy plenáris ülésre, délután elkülönített szekcióülésekre, a plenáris ülés végén pedig egy kerekasztal-beszélgetésre került sor a munkaerőpiac igényeinek változásával kapcsolatban. Ezen a beszélgetésen több hazai vállalat vezetője mellett egyetemünket Trautmann László dékán úr és Hofmeister Ágnes dékán asszony képviselte. A plenáris ülésen először Mészáros Tamás rektor úr előadását hallgathattuk meg. Rektor úr előrebocsátotta, hogy előzetes egyeztetés alapján ő az egyetemi stratégiáról fog beszélni, és Trautmann dékán úrra hagyja a kari stratégia taglalását.

Animal models of group-B streptococcal infections are needed to develop effective therapies. Here, Andrade et al. present a mouse model in which the bacteria are transmitted from vaginally colonised pregnant females to their offspring, causing neonatal meningitis and neurological developmental disabilities.

Sweetpotato yield depends on a change in the developmental fate of adventitious roots into storage-roots. The mechanisms underlying this developmental switch are still unclear. We examined the hypothesis claiming that regulation of root lignification determines storage-root formation. We show that application of the plant hormone gibberellin increased stem elongation and root gibberellin levels, while having inhibitory effects on root system parameters, decreasing lateral root number and length, and significantly reducing storage-root number and diameter. Furthermore, gibberellin enhanced root xylem development, caused increased lignin deposition, and, at the same time, decreased root starch accumulation. In accordance with these developmental effects, gibberellin application upregulated expression levels of sweetpotato orthologues of Arabidopsis vascular development regulators (IbNA075, IbVND7, and IbSND2) and of lignin biosynthesis genes (IbPAL, IbC4H, Ib4CL, IbCCoAOMT, and IbCAD), while downregulating starch biosynthesis genes (IbAGPase and IbGBSS) in the roots. Interestingly, gibberellin downregulated root expression levels of orthologues of the Arabidopsis BREVIPEDICELLUS transcription factor (IbKN2 and IbKN3), regulator of meristem maintenance. The results substantiate our hypothesis and mark gibberellin as an important player in regulation of sweetpotato root development, suggesting that increased fiber formation and lignification inhibit storage-root formation and yield. Taken together, our findings provide insight into the mechanisms underlying sweetpotato storage-root formation and provide a valuable database of genes for further research.

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.