Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

Blocking Neuronal Signaling to Immune Cells Treats Streptococcal Invasive Infection.

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.

Induction of Autonomous Memory Alveolar Macrophages Requires T Cell Help and Is Critical to Trained Immunity.

Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.

CD4+ T cell-mediated recognition of a conserved cholesterol-dependent cytolysin epitope generates broad antibacterial immunity.

CD4+ T cell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and invasive pneumococcal diseases (IPDs). Although such immune responses are common, the pertinent antigens have remained elusive. We identified an immunodominant CD4+ T cell epitope derived from pneumolysin (Ply), a member of the bacterial cholesterol-dependent cytolysins (CDCs). This epitope was broadly immunogenic as a consequence of presentation by the pervasive human leukocyte antigen (HLA) allotypes DPB1∗02 and DPB1∗04 and recognition via architecturally diverse T cell receptors (TCRs). Moreover, the immunogenicity of Ply427-444 was underpinned by core residues in the conserved undecapeptide region (ECTGLAWEWWR), enabling cross-recognition of heterologous bacterial pathogens expressing CDCs. Molecular studies further showed that HLA-DP4-Ply427-441 was engaged similarly by private and public TCRs. Collectively, these findings reveal the mechanistic determinants of near-global immune focusing on a trans-phyla bacterial epitope, which could inform ancillary strategies to combat various life-threatening infectious diseases, including IPDs.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.

YkuR functions as a protein deacetylase in Streptococcus mutans.

Protein acetylation is a common and reversible posttranslational modification tightly governed by protein acetyltransferases and deacetylases crucial for various biological processes in both eukaryotes and prokaryotes. Although recent studies have characterized many acetyltransferases in diverse bacterial species, only a few protein deacetylases have been identified in prokaryotes, perhaps in part due to their limited sequence homology. In this study, we identified YkuR, encoded by smu_318, as a unique protein deacetylase in Streptococcus mutans. Through protein acetylome analysis, we demonstrated that the deletion of ykuR significantly upregulated protein acetylation levels, affecting key enzymes in translation processes and metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, and biofilm formation. In particular, YkuR modulated extracellular polysaccharide synthesis and biofilm formation through the direct deacetylation of glucosyltransferases (Gtfs) in the presence of NAD+. Intriguingly, YkuR can be acetylated in a nonenzymatic manner, which then negatively regulated its deacetylase activity, suggesting the presence of a self-regulatory mechanism. Moreover, in vivo studies further demonstrated that the deletion of ykuR attenuated the cariogenicity of S. mutans in the rat caries model, substantiating its involvement in the pathogenesis of dental caries. Therefore, our study revealed a unique regulatory mechanism mediated by YkuR through protein deacetylation that regulates the physiology and pathogenicity of S. mutans.

IL-27 regulates the differentiation of follicular helper NKT cells via metabolic adaptation of mitochondria.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Quantification of Adaptive Immune Responses Against Protein-Binding Interfaces in the Streptococcal M1 Protein.

Bacterial or viral antigens can contain subdominant protein regions that elicit weak antibody responses upon vaccination or infection although there is accumulating evidence that antibody responses against subdominant regions can enhance the protective immune response. One proposed mechanism for subdominant protein regions is the binding of host proteins that prevent antibody production against epitopes hidden within the protein binding interfaces. Here, we used affinity purification combined with quantitative mass spectrometry (AP-MS) to examine the level of competition between antigen-specific antibodies and host-pathogen protein interaction networks using the M1 protein from Streptococcus pyogenes as a model system. As most humans have circulating antibodies against the M1 protein, we first used AP-MS to show that the M1 protein interspecies protein network formed with human plasma proteins is largely conserved in naïve mice. Immunizing mice with the M1 protein generated a time-dependent increase of anti-M1 antibodies. AP-MS analysis comparing the composition of the M1-plasma protein network from naïve and immunized mice showed significant enrichment of 292 IgG peptides associated with 56 IgG chains in the immune mice. Despite the significant increase of bound IgGs, the levels of interacting plasma proteins were not significantly reduced in the immune mice. The results indicate that the antigen-specific polyclonal IgG against the M1 protein primarily targets epitopes outside the other plasma protein binding interfaces. In conclusion, this study demonstrates that AP-MS is a promising strategy to determine the relationship between antigen-specific antibodies and host-pathogen interaction networks that could be used to define subdominant protein regions of relevance for vaccine development.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme-iron from human hemoglobin.

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's ß subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells.

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell-cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein-1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway.

Membrane vesicle delivery of a streptococcal M protein disrupts the blood-brain barrier by inducing autophagic endothelial cell death.

M family proteins are critical virulence determinants of Streptococci. Streptococcus equi subsp. zooepidemicus (SEZ) are Group C streptococci that cause meningitis in animals and humans. SzM, the M protein of SEZ, has been linked to SEZ brain invasion. Here, we demonstrate that SzM is important in SEZ disruption of the blood-brain barrier (BBB). SEZ release SzM-bound membrane vesicles (MVs), and endocytosis of these vesicles by human brain endothelial microvascular cells (hBMECs) results in SzM-dependent cytotoxicity. Furthermore, administration of SzM-bound MVs disrupted the murine BBB. A CRISPR screen revealed that SzM cytotoxicity in hBMECs depends on PTEN-related activation of autophagic cell death. Pharmacologic inhibition of PTEN activity prevented SEZ disruption of the murine BBB and delayed mortality. Our data show that MV delivery of SzM to host cells plays a key role in SEZ pathogenicity and suggests that MV delivery of streptococcal M family proteins is likely a common streptococcal virulence mechanism.

Streptococcus dysgalactiae subsp. equisimilis infection and its intersection with Streptococcus pyogenes.

SUMMARYStreptococcus dysgalactiae subsp. equisimilis (SDSE) is an increasingly recognized cause of disease in humans. Disease manifestations range from non-invasive superficial skin and soft tissue infections to life-threatening streptococcal toxic shock syndrome and necrotizing fasciitis. Invasive disease is usually associated with co-morbidities, immunosuppression, and advancing age. The crude incidence of invasive disease approaches that of the closely related pathogen, Streptococcus pyogenes. Genomic epidemiology using whole-genome sequencing has revealed important insights into global SDSE population dynamics including emerging lineages and spread of anti-microbial resistance. It has also complemented observations of overlapping pathobiology between SDSE and S. pyogenes, including shared virulence factors and mobile gene content, potentially underlying shared pathogen phenotypes. This review provides an overview of the clinical and genomic epidemiology, disease manifestations, treatment, and virulence determinants of human infections with SDSE with a particular focus on its overlap with S. pyogenes. In doing so, we highlight the importance of understanding the overlap of SDSE and S. pyogenes to inform surveillance and disease control strategies.

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Ancient Genomes From Bronze Age Remains Reveal Deep Diversity and Recent Adaptive Episodes for Human Oral Pathobionts.

Ancient microbial genomes can illuminate pathobiont evolution across millenia, with teeth providing a rich substrate. However, the characterization of prehistoric oral pathobiont diversity is limited. In Europe, only preagricultural genomes have been subject to phylogenetic analysis, with none compared to more recent archaeological periods. Here, we report well-preserved microbiomes from two 4,000-year-old teeth from an Irish limestone cave. These contained bacteria implicated in periodontitis, as well as Streptococcus mutans, the major cause of caries and rare in the ancient genomic record. Despite deriving from the same individual, these teeth produced divergent Tannerella forsythia genomes, indicating higher levels of strain diversity in prehistoric populations. We find evidence of microbiome dysbiosis, with a disproportionate quantity of S. mutans sequences relative to other oral streptococci. This high abundance allowed for metagenomic assembly, resulting in its first reported ancient genome. Phylogenetic analysis indicates major postmedieval population expansions for both species, highlighting the inordinate impact of recent dietary changes. In T. forsythia, this expansion is associated with the replacement of older lineages, possibly reflecting a genome-wide selective sweep. Accordingly, we see dramatic changes in T. forsythia's virulence repertoire across this period. S. mutans shows a contrasting pattern, with deeply divergent lineages persisting in modern populations. This may be due to its highly recombining nature, allowing for maintenance of diversity through selective episodes. Nonetheless, an explosion in recent coalescences and significantly shorter branch lengths separating bacteriocin-carrying strains indicate major changes in S. mutans demography and function coinciding with sugar popularization during the industrial period.