ABSTRACT Plasmalogen is a specific glycerophospholipid present in both animal and bacterial organisms. It plays a crucial function in eukaryotic cellular processes and is closely related to several human diseases, including neurological disorders and cancers. Nonetheless, the precise biological role of plasmalogen in bacteria is not well understood. In this study, we identified SMU_438c as the enzyme responsible for plasmalogen production in Streptococcus mutans under anaerobic conditions. The heterologous expression of SMU_438c in a plasmalogen-negative strain, Streptococcus sanguinis , resulted in the production of plasmalogen, indicating that this enzyme is sufficient for plasmalogen production. Additionally, the plasmalogen-deficient S. mutans exhibited significantly lower acid tolerance and diminished its colonization in Drosophila flies compared to the wild-type strain and complemented strain. In summary, our data suggest that plasmalogen plays a vital role in bacterial stress tolerance and in vivo colonization. IMPORTANCE This study sheds light on the biological role of plasmalogen, a specific glycerophospholipid, in bacteria, particularly in Streptococcus mutans . Plasmalogens are known for their significant roles in eukaryotic cells and have been linked to human diseases like neurological disorders and cancers. The enzyme SMU_438c, identified as essential for plasmalogen production under anaerobic conditions, was crucial for acid tolerance and in vivo colonization in Drosophila by S. mutans , underscoring its importance in bacterial stress response and colonization. These findings bridge the knowledge gap in bacterial physiology, highlighting plasmalogen’s role in microbial survival and offering potential insights into microbial pathogenesis and host–microbe interactions.

Streptococcal pyruvate oxidase (SpxB) is a hydrogen peroxide-generating enzyme and plays a critical role in Streptococcus sanguinis interspecies interactions, but less is known about its biochemistry. We examined SpxB subcellular localization using protein fractionation and microscopy and found SpxB to be primarily cytoplasmic, but a small portion is also membrane associated. Potential post-translational modifications of SpxB were determined using coimmunoprecipitation and mass spectrometry. Two mutant strains were constructed to further validate the presence of predicted site-specific post-translational modifications. These site mutated SpxB proteins exhibited reduced solubility in vivo, which likely contributes to the observed phenotypic changes in colony morphology, bacterial growth, and H2O2 production. Overall, our data suggest that SpxB post-translational modifications likely play a major role to regulate SpxB function in S. sanguinis.

Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis.

Regulated RNA turnover is vital for the control of gene expression in all cellular life. In Escherichia coli, this process is largely controlled by a stable degradosome complex containing RNase E and a variety of additional enzymes. In the Firmicutes phylum, species lack RNase E and often encode the paralogous enzymes RNase J1 and RNase J2. Unlike RNase J1, surprisingly little is known about the regulatory function and protein interactions of RNase J2, despite being a central pleiotropic regulator for the streptococci and other closely related organisms. Using crosslink coimmunoprecipitation in Streptococcus mutans, we have identified the major proteins found within RNase J2 protein complexes located in the cytoplasm and at the cell membrane. In both subcellular fractions, RNase J2 exhibited the most robust interactions with RNase J1, while additional transient and/or weaker “degradosome-like” interactions were also detected. In addition, RNase J2 exhibits multiple novel interactions that have not been previously reported for any RNase J proteins, some of which were highly biased for either the cytoplasmic or membrane fractions. We also determined that the RNase J2 C-terminal domain (CTD) encodes a structure that is likely conserved among RNase J enzymes and may have an analogous function to the C-terminal portion of RNase E. While we did observe a number of parallels between the RNase J2 interactome and the E. coli degradosome paradigm, our results suggest that S. mutans degradosomes are either unlikely to exist or are quite distinct from those of E. coli.

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) is often a commensal bacterium that colonizes healthy adults asymptomatically and is a frequent inhabitant of the vaginal tract in women. However, in immunocompromised individuals, particularly the newborn, GBS may transition to an invasive pathogen and cause serious disease. Despite the use of the currently recommended intrapartum antibiotic prophylaxis for GBS-positive mothers, GBS remains a leading cause of neonatal septicemia and meningitis. To adapt to the various host environments encountered during its disease cycle, GBS possesses multiple two-component regulatory systems (TCSs). Here we investigated the contribution of a transcriptional regulator containing a LytTR domain, LtdR, to GBS pathogenesis. Disruption of the ltdR gene in the GBS chromosome resulted in a significant increase in bacterial invasion into human cerebral microvascular endothelial cells (hCMEC) in vitro as well as the greater penetration of the blood-brain barrier (BBB) and the development of meningitis in vivo . Correspondingly, infection of hCMEC with the Δ ltdR mutant resulted in increased secretion of the proinflammatory cytokines interleukin-8 (IL-8), CXCL-1, and IL-6. Further, using a mouse model of GBS vaginal colonization, we observed that the Δ ltdR mutant was cleared more readily from the vaginal tract and also that infection with the Δ ltdR mutant resulted in increased cytokine production from human vaginal epithelial cells. RNA sequencing revealed global transcriptional differences between the Δ ltdR mutant and the parental wild-type GBS strain. These results suggest that LtdR regulates many bacterial processes that can influence GBS-host interactions to promote both bacterial persistence and disease progression.

Background: Group B Streptococcus (GBS) is a major cause of invasive disease especially in neonates. In GBS three structurally distinct pilus polymers have been identified as important virulence factors and promising vaccine candidates. The vast majority of Group B Streptococci belonging to the hypervirulent serotype III ST-17 lineage bear pilus types 1 and 2b. The purpose of this study was to investigate the relative contribution of these two pilus types to the pathogenesis of a ST-17 strain.Results: We performed in vivo and in vitro analysis of isogenic knockout mutants derived from the GBS COH1 ST-17 strain deprived of either pilus type 1 or 2b. We compared the two pilus mutants with the wild type strain in a mouse model of invasive disease, in vitro survival in macrophages, and adherence/ invasion assays using human brain endothelial and lung epithelial cell lines. Significantly less of the pilus 2b mutant was recovered from the blood, lungs and brain tissue of infected mice compared to the wild-type and pilus 1 mutant strains. Further, while the pilus 2b mutant survived similarly in murine macrophages, it exhibited a lower capacity to adhere and invade human brain epithelial and lung endothelial cell lines.Conclusions: The data suggest an important role of pilus 2b in mediating GBS infection and host cell interaction of strains belonging to the hypervirulent GBS ST-17 lineage.

Group B Streptococcus (GBS) is a major causative agent of neonatal meningitis due to its ability to efficiently cross the blood-brain barrier (BBB) and enter the central nervous system (CNS). It has been demonstrated that GBS can invade human brain microvascular endothelial cells (hBMEC), a primary component of the BBB; however, the mechanism of intracellular survival and trafficking is unclear. We previously identified a two component regulatory system, CiaR/H, which promotes GBS intracellular survival in hBMEC. Here we show that a GBS strain deficient in the response regulator, CiaR, localized more frequently with Rab5, Rab7 and LAMP1 positive vesicles. Further, lysosomes isolated from hBMEC contained fewer viable bacteria following initial infection with the ΔciaR mutant compared to the WT strain. To characterize the contribution of CiaR-regulated genes, we constructed isogenic mutant strains lacking the two most down-regulated genes in the CiaR-deficient mutant, SAN_2180 and SAN_0039. These genes contributed to bacterial uptake and intracellular survival. Furthermore, competition experiments in mice showed that WT GBS had a significant survival advantage over the Δ2180 and Δ0039 mutants in the bloodstream and brain.

ABSTRACT Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence. IMPORTANCE Two-component systems have been evolved by bacteria to detect environmental changes, and they play key roles in pathogenicity. A comprehensive analysis of TCS in GBS has not been performed previously. In this work, we classify 21 TCS and present evidence for the involvement of two specific TCS in GBS virulence and colonization in vivo. Although pinpointing specific TCS stimuli is notoriously difficult, we used a combination of techniques to identify two systems with different effects on GBS pathogenesis. For one of the systems, we propose that fructose-6-phosphate, a metabolite in glycolysis, is sufficient to induce a regulatory response involving a sugar transport system. Our catalogue and classification of TCS may guide further studies into the role of TCS in GBS pathogenicity and biology.

ABSTRACT Group B Streptococcus (GBS) is currently the leading cause of neonatal meningitis. This is due to its ability to survive and multiply in the bloodstream and interact with specialized human brain microvascular endothelial cells (hBMEC), which constitute the blood-brain barrier (BBB). The exact mechanism(s) of GBS-BBB penetration is still largely unknown. We and others have shown that GBS interacts with components of the extracellular matrix. In this study, we demonstrate that GBS of representative serotypes binds immobilized and cell surface fibronectin and identify a putative fibronectin binding protein, s treptococcal f ibronectin b inding protein A (SfbA). Allelic replacement of sfbA in the GBS chromosome resulted in a significant decrease in ability to bind fibronection and invade hBMEC compared with the wild-type (WT) parental strain. Expression of SfbA in the noninvasive strain Lactococcus lactis was sufficient to promote fibronectin binding and hBMEC invasion. Furthermore, the addition of an antifibronectin antibody or an RGD peptide that blocks fibronectin binding to integrins significantly reduced invasion of the WT but not the sfbA -deficient mutant strain, demonstrating the importance of an SfbA-fibronectin-integrin interaction for GBS cellular invasion. Using a murine model of GBS meningitis, we also observed that WT GBS penetrated the brain and established meningitis more frequently than did the Δ sfbA mutant strain. Our data suggest that GBS SfbA plays an important role in bacterial interaction with BBB endothelium and the pathogenesis of streptococcal meningitis.

Background: Penetration of brain endothelium by Group B streptococcus (GBS) is the first step in the development of meningitis. Results: Autophagy is activated in response to GBS infection. Conclusion: Autophagy induction occurs through GBS toxin expression, while key autophagic proteins contribute to GBS destruction. Significance: Understanding the role of autophagy in brain endothelium may inform novel strategies to prevent the pathogenesis of bacterial meningitis. Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.

The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial cells (hBMEC), the molecular basis for this interaction is not well defined. We now demonstrate that Srr1 contributes to GBS attachment to hBMEC via the direct interaction of its binding region (BR) with human fibrinogen. When assessed by Far Western blotting, Srr1 was the only protein in GBS extracts that bound fibrinogen. Studies using recombinant Srr1-BR and purified fibrinogen in vitro confirmed a direct protein-protein interaction. Srr1-BR binding was localized to amino acids 283–410 of the fibrinogen Aα chain. Structural predictions indicated that the conformation of Srr1-BR is likely to resemble that of SdrG and other related staphylococcal proteins that bind to fibrinogen through a “dock, lock, and latch” mechanism (DLL). Deletion of the predicted latch domain of Srr1-BR abolished the interaction of the BR with fibrinogen. In addition, a mutant GBS strain lacking the latch domain exhibited reduced binding to hBMEC, and was significantly attenuated in an in vivo model of meningitis. These results indicate that Srr1 can bind fibrinogen directly likely through a DLL mechanism, which has not been described for other streptococcal adhesins. This interaction was important for the pathogenesis of GBS central nervous system invasion and subsequent disease progression.

The interaction of the filamentous fungal plant pathogen Cryphonectria parasitica with its virulence-attenuating viruses provides a unique platform to explore the molecular biology and genetics of virus-host interactions. Following the development of transformation procedures for this fungus, subsequent advances include infectious cDNA clones of several members of the Hypoviridae and an imminently complete fungal genome project. Presented here are basic protocols for growth of the organism and the extraction of DNA, RNA, and protein. Additionally, two further protocols are provided for investigations of host protein phosphorylation and for viral genome secondary structure.

Virulence-attenuating viruses (hypoviruses) of the filamentous fungus Cryphonectria parasitica, the causative agent of chestnut blight, have become a premier model for understanding the molecular biology of mycoviruses. However, a major gap exists in current understanding of structure and function of the untranslated regions (UTRs) of the hypovirus RNA genome, despite considerable evidence that secondary and tertiary UTR structure plays a crucial role in the control of translation and genome replication in other systems. In this study we have used structure prediction software coupled with RNase digestion studies to develop validated structural models for the 5′ UTRs of the two best-characterized members of the Hypoviridae, CHV1-EP713 and CHV1-Euro7. These two hypovirus strains exhibit significant variation in virulence attenuation despite sharing >90% sequence identity. Our models reveal highly structured regions in the 5′ UTR of both strains, with numerous stem-loops suggestive of internal ribosome entry sites. However, considerable differences in the size and complexity of structural elements exist between the two strains. These data will guide future, mutagenesis-based studies of the structural requirements for hypovirus genome replication and translation.