Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

Adapting the endoplasmic reticulum proteostasis rescues epilepsy-associated NMDA receptor variants.

The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired function on neuronal membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we investigated the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A subunit, including M705V and A727T. In the transfected HEK293T cells, we showed that the two variants were targeted to the proteasome for degradation and had reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (binding immunoglobulin protein) in the endoplasmic reticulum (ER), dose-dependently enhanced the functional surface expression of the M705V and A727T variants in HEK293T cells. Moreover, BIX (10 µM) increased the surface protein levels of the M705V variant in human iPSC-derived neurons. We revealed that BIX promoted folding, inhibited degradation, and enhanced anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results suggest that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction.

S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates.

Streptococcus pneumoniae is capable of randomly switching their genomic DNA methylation pattern between six distinct bacterial subpopulations (A-F) via recombination of a type 1 restriction-modification locus, spnIII. These pneumococcal subpopulations exhibit phenotypic changes which favor carriage or invasive disease. In particular, the spnIIIB allele has been associated with increased nasopharyngeal carriage and the downregulation of the luxS gene. The LuxS/AI-2 QS system represent a universal language for bacteria and has been linked to virulence and biofilm formation in S. pneumoniae. In this work, we have explored the link between spnIII alleles, the luxS gene and virulence in two clinical pneumococcal isolates from the blood and cerebrospinal fluid (CSF) of one pediatric meningitis patient. The blood and CSF strains showed different virulence profiles in mice. Analysis of the spnIII system of these strains recovered from the murine nasopharynx showed that the system switched to different alleles commensurate with the initial source of the isolate. Of note, the blood strain showed high expression of spnIIIB allele, previously linked with less LuxS protein production. Importantly, strains with deleted luxS displayed different phenotypic profiles compared to the wildtype, but similar to the strains recovered from the nasopharynx of infected mice. This study used clinically relevant S. pneumoniae strains to demonstrate that the regulatory network between luxS and the type 1 restriction-modification system play a key role in infections and may support different adaptation to specific host niches.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

A selfish bacteriocin dictates pneumococcal domination.

Efficient colonization of new hosts is critical for survival of Streptococcus pneumoniae. In this issue of Cell Host & Microbe, Aggarwal et al. investigate the population dynamics of this process, demonstrating how a quorum-sensing bacteriocin locus promotes survival of individual clonal lineages, providing a population bottleneck and restricting genetic diversity.

Serum Neu5Gc biomarkers are elevated in primary cutaneous melanoma.

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.

Simple and practical sialoglycan encoding system reveals vast diversity in nature and identifies a universal sialoglycan-recognizing probe derived from AB5 toxin B subunits.

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N-glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

A Nonadjuvanted Whole-Inactivated Pneumococcal Vaccine Induces Multiserotype Opsonophagocytic Responses Mediated by Noncapsule-Specific Antibodies.

Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children <5 years of age every single year, and is the leading cause of pneumonia-associated mortality globally. While the capsular polysaccharide (CPS)-based vaccine Prevnar13 prevents serious illness caused by 13 serotypes, ongoing Prevnar13 use has driven the emergence of nonincluded serotypes as major causes of infection and disease. To overcome this issue, we have developed a next-generation pneumococcal vaccine conferring serotype-independent protection. This vaccine shows equivalent or superior efficacy to Prevnar13, and performance was heightened when our vaccine was administered with no adjuvant. These findings should be considered for similar vaccines in development, as the benefit of adjuvant is often assumed and its automatic inclusion may be limiting product efficacy, resulting in potential abandonment of viable vaccine candidates, or prolonging their time to clinic.

A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.

Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

CONTEXT: Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. OBJECTIVE: We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and ß-cell function. METHODS: Human islets were treated with interleukin-1ß and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. RESULTS: Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. CONCLUSION: Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate ß-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

Ca2+ signalling system initiated by endoplasmic reticulum stress stimulates PERK activation.

The accumulation of unfolded proteins within the Endoplasmic Reticulum (ER) activates a signal transduction pathway termed the unfolded protein response (UPR), which attempts to restore ER homoeostasis. If this cannot be done, UPR signalling ultimately induces apoptosis. Ca2+ depletion in the ER is a potent inducer of ER stress. Despite the ubiquity of Ca2+ as an intracellular messenger, the precise mechanism(s) by which Ca2+ release affects the UPR remains unknown. Tethering a genetically encoded Ca2+ indicator (GCamP6) to the ER membrane revealed novel Ca2+ signalling events initiated by Ca2+ microdomains in human astrocytes under ER stress, induced by tunicamycin (Tm), an N-glycosylation inhibitor, as well as in a cell model deficient in all three inositol triphosphate receptor isoforms. Pharmacological and molecular studies indicate that these local events are mediated by translocons and that the Ca2+ microdomains impact (PKR)-like-ER kinase (PERK), an UPR sensor, activation. These findings reveal the existence of a Ca2+ signal mechanism by which stressor-mediated Ca2+ release regulates ER stress.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis.

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community.

Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP.

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.

Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression.

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes-a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. IMPORTANCE S. pneumoniae is the world's foremost bacterial pathogen. S. pneumoniae encodes a phasevarion (phase-variable regulon), that results in differential expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes.

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infected mice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits.

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

The Role of luxS in the Middle Ear Streptococcus pneumoniae Isolate 947.

The LuxS protein, encoded by luxS, is required for the production of autoinducer 2 (AI-2) in Streptococcus pneumoniae. The AI-2 molecule serves as a quorum sensing signal, and thus regulates cellular processes such as carbohydrate utilisation and biofilm formation, as well as impacting virulence. The role of luxS in S. pneumoniae biology and lifestyle has been predominantly assessed in the laboratory strain D39. However, as biofilm formation, which is regulated by luxS, is critical for the ability of S. pneumoniae to cause otitis media, we investigated the role of luxS in a middle ear isolate, strain 947. Our results identified luxS to have a role in prevention of S. pneumoniae transition from colonisation of the nasopharynx to the ear, and in facilitating adherence to host epithelial cells.