Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

The long noncoding RNA ROCKI regulates inflammatory gene expression.

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI-APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.

Homeostasis of cellular amino acids in Acanthamoeba castellanii exposed to different media under amoeba-bacteria coculture conditions.

BACKGROUND: Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli. METHOD: A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii. RESULTS: Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine. DISCUSSION: Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked. CONCLUSION: When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.

Harnessing antifungal immunity in pursuit of a Staphylococcus aureus vaccine strategy.

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.

Potential role of Acanthamoeba Rab7.

Acanthamoeba castellanii is a free-living protozoan that causes several severe human parasitic diseases such as Acanthamoeba keratitis and granulomatous encephalitis. A. castellanii feeds on bacteria, yeasts, and other organic particles as food sources, but the mechanisms of digestion in acanthamoebal cells are unclear. Rab GTPases participate in endosomal delivery in eukaryotes after phagocytosis. This study aimed to determine the potential functions of A. castellanii Rab7 (AcRab7), which is involved in phagocytosis, and the relationship between AcRab7 and further cellular physiological phenomena. In this study, the inhibitor CID1067700 (CID) was used to specifically inhibit the binding of nucleotides to confirm the potential functions of AcRab7. Cellular proliferation and ATP assays were also used to detect underlying cellular physiological functions after blocking the phagocytosis pathway. We found that AcRab7 expression increased as the co-culture time with Escherichia coli increased. Immunofluorescence staining showed that AcRab7 colocalized with lysosomes in its GTP-activating form. In addition, AcRab7 inhibition resulted in a reduction in cell proliferation and ATP levels. Our results suggest that AcRab7 participates in endosomal delivery and dominates energy production and cell growth.

Adoptive Transfer of Serum Samples From Children With Invasive Staphylococcal Infection and Protection Against Staphylococcus aureus Sepsis.

A successful Staphylococcus aureus vaccine remains elusive, and one controversy in the field is whether humans generate a protective adaptive immune response to infection. We developed a bacterial challenge murine assay that directly assesses the protective capacity of adoptively transferred human serum samples. We first validated the model by showing that postpneumococcal vaccine serum samples from humans induced effective clearance of Streptococcus pneumoniae in mice. We then found that human serum samples adoptively transferred from children with invasive S. aureus infections exhibited protection from disease in a murine model, with some samples conferring near complete protection. These findings demonstrate that human serum samples are capable of conferring a protective adaptive response generated by humans during invasive staphylococcal disease, allowing for the study of protective factors in a murine model. Identification of the protective factors present in the most efficacious serum samples would be of high interest as potential staphylococcal vaccine candidates or passive therapeutics.

Siglec-7 engagement by GBS ß-protein suppresses pyroptotic cell death of natural killer cells.

Natural killer (NK) cells are innate immune lymphocytes that recognize and destroy abnormal host cells, such as tumor cells or those infected by viral pathogens. To safely accomplish these functions, NK cells display activating receptors that detect stress molecules or viral ligands displayed at the cell surface, balanced by inhibitory receptors that bind to self-molecules. To date, such activating and inhibitory receptors on NK cells are not known to recognize bacterial determinants. Moreover, NK cell responses to direct interactions with extracellular bacteria are poorly explored. In this study, we observed the human neonatal pathogen group B Streptococcus (GBS) can directly engage human NK cells. The interaction was mediated through the B6N segment of streptococcal ß-protein, binding to the inhibitory receptor Siglec-7 via its amino-terminal V-set domain. Unlike classical Siglec binding, the interaction is also independent of its sialic acid recognition property. In contrast to WT GBS, mutants lacking ß-protein induced efficient pyroptosis of NK cells through the NLRP3 inflammasome, with production and secretion of the proinflammatory cytokine IL-1ß and dissemination of the cytotoxic molecule granzyme B. We postulate that GBS evolved ß-protein engagement of inhibitory human Siglec-7 to suppress the pyroptotic response of NK cells and thereby block recruitment of a broader innate immune response, i.e., by "silencing the sentinel."

Effects of Acanthamoeba castellanii on the dissolved oxygen and the microbial community under the experimental aquatic model.

Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

The characteristics of pre-existing humoral imprint determine efficacy of S. aureus vaccines and support alternative vaccine approaches.

The failure of the Staphylococcus aureus (SA) IsdB vaccine trial can be explained by the recall of non-protective immune imprints from prior SA exposure. Here, we investigate natural human SA humoral imprints to understand their broader impact on SA immunizations. We show that antibody responses against SA cell-wall-associated antigens (CWAs) are non-opsonic, while antibodies against SA toxins are neutralizing. Importantly, the protective characteristics of the antibody imprints accurately predict the failure of corresponding vaccines against CWAs and support vaccination against toxins. In passive immunization platforms, natural anti-SA human antibodies reduce the efficacy of the human monoclonal antibodies suvratoxumab and tefibazumab, consistent with the results of their respective clinical trials. Strikingly, in the absence of specific humoral memory responses, active immunizations are efficacious in both naive and SA-experienced mice. Overall, our study points to a practical and predictive approach to evaluate and develop SA vaccines based on pre-existing humoral imprint characteristics.

Detection of trichomonads in patients with lung cancer and transcription analysis on the response of human pulmonary epithelial cells to Trichomonas tenax invasion.

INTRODUCTION: Lung cancer is one of the most prevalent malignancies worldwide. Substantial research has illuminated the intricate interplay between microorganisms and human health, revealing their role in disease regulation. Trichomonads is a flagellated protozoan in the human cavity and have been previously identified as a pathogen associated with pneumonia, contributing to tissue chronic inflammation and carcinogenesis. METHODS: Nested polymerase chain reaction methods were employed to scrutinize the prevalence of trichomonads in the bronchovesicular fluid of patients diagnosed with lung cancer. Subsequently, the influence of Trichomonas tenax invasion on lung cancer cells was elucidated through proliferation assays, migration assays, and transcription analysis. RESULTS: Bronchoalveolar fluid samples from lung cancer patients yielded positive nested PCR results for eight out of twenty-seven samples. Seven of these samples were identified as Trichomonas tenax, while one was identified as Tetratrichomonas spp. Our findings revealed a significant upregulation of pathways associated with carcinogenesis, including cellular proliferation, migration, and drug resistance, in response to T. tenax invasion. CONCLUSIONS: This study underscores the importance of recognizing the presence of trichomonads and the influence of T. tenax invasion on host responses to respiratory diseases. The identified pathways implicated in cancer development may pave the way for developing targeted treatment strategies for pulmonary diseases. These findings hold promise for informing and improving the precision of therapeutic interventions in the context of pulmonary ailments.

TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade.

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

Advanced mass spectrometry and chemical analyses reveal the presence of terminal disialyl motif on mouse B-cell glycoproteins.

The occurrence of a terminal disialyl motif on mammalian O-glycans is increasingly being identified through recent mass spectrometry (MS)-based glycomic profiling. In most cases, it is carried on simple core 1 structures in which both the galactose and N-acetyl galactosamine can be disialylated. In contrast, a disialyl motif on N-glycans is less readily revealed by MS mapping, since additional MS/MS analysis is required to determine the distribution of the various sialic acids on typically multisialylated complex type N-glycans. In our MS-based glycomic screening, we found that a mouse B lymphoma cell line, BCL1, ranks among those that have the highest amount of disialyl motif on its O-glycans, including those carried on CD45. More intriguingly, detailed chemical and MS/MS analyses unambiguously showed that the Neu5Gcα2-8Neu5Gc disialyl motif is also present on the N-glycans and that it can be carried on the termini of polylactosaminoglycan chains, which can be further sulfated on the proximal GlcNAc, occurring alongside other monosialylated sulfated LacNAc termini. Upon silencing the expression of mouse α2,8-sialyltransferase VI (ST8Sia VI), the overall disialyl content decreases significantly, but more so for that on the N-glycans than the O-glycans. ST8Sia VI was further shown to be the most significantly upregulated ST8Sia during plasma cell differentiation, which coincides with increasing content of the disialyl motif. Increasing terminal disialylation without leading to polysialylation may thus have important biological consequences awaiting further investigation. Likewise, the expression of mono- and disialylated sulfated LacNAc may constitute novel recognition codes modulating B-cell activation and differentiation.

Galectin-1 and galectin-8 have redundant roles in promoting plasma cell formation.

Galectin (Gal) family members are a type of soluble lectin, and they play important roles in immunomodulation. Their redundant roles have been proposed. We previously found that Gal-1 promotes the formation of Ab-secreting plasma cells, but B cells from Gal-1-deficient and control animals produce comparable amounts of Abs. In the current study, we used synthetic sulfomodified N-acetyllactosamine (LacNAc) analogs and short hairpin RNAs for Gal-8 to demonstrate a redundancy in the effects of Gal-1 and Gal-8 on plasma cell formation. Gal-1 and Gal-8 were both expressed during plasma cell differentiation, and both Gals promoted the formation of plasma cells. Gal-1 and Gal-8 bound better to mature B cells than to plasma cells, and the expression of glycosyltransferase enzymes changed during differentiation, with a decrease in mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetyl-glucosaminyltransferase and N-acetylglucosaminyltransferase-1 mRNAs in plasma cells. Synthetic sulfomodified Galß1-3GlcNAc disaccharides (type 1 LacNAcs) selectively prevented Gal-8 binding, leading to a blockade of Ab production in Gal-1-deficient B cells. Furthermore, synthetic type 1 LacNAcs that were able to block the binding of both Gals greatly reduced the effect of exogenously added recombinant Gal-1 and Gal-8 on promoting Ab production. These results reveal a novel role for Gal-8 in collaboration with Gal-1 in plasma cell formation, and suggest the possibility of using distinct LacNAc ligands to modulate the function of Gals.

Functional divergence of a bacterial enzyme promotes healthy or acneic skin.

Acne is a dermatologic disease with a strong pathologic association with human commensal Cutibacterium acnes. Conspicuously, certain C. acnes phylotypes are associated with acne, whereas others are associated with healthy skin. Here we investigate if the evolution of a C. acnes enzyme contributes to health or acne. Two hyaluronidase variants exclusively expressed by C. acnes strains, HylA and HylB, demonstrate remarkable clinical correlation with acne or health. We show that HylA is strongly pro-inflammatory, and HylB is modestly anti-inflammatory in a murine (female) acne model. Structural and phylogenic studies suggest that the enzymes evolved from a common hyaluronidase that acquired distinct enzymatic activity. Health-associated HylB degrades hyaluronic acid (HA) exclusively to HA disaccharides leading to reduced inflammation, whereas HylA generates large-sized HA fragments that drive robust TLR2-dependent pathology. Replacing an amino acid, Serine to Glycine near the HylA catalytic site enhances the enzymatic activity of HylA and produces an HA degradation pattern intermediate to HylA and HylB. Selective targeting of HylA using peptide vaccine or inhibitors alleviates acne pathology. We suggest that the functional divergence of HylA and HylB is a major driving force behind C. acnes health- and acne- phenotype and propose targeting of HylA as an approach for acne therapy.

Acyl and silyl group effects in reactivity-based one-pot glycosylation: synthesis of embryonic stem cell surface carbohydrates Lc4 and IV(2)Fuc-Lc4.

Relative reactivity evaluations showed the graded arming of toluenyl thioglucosides by variously positioned silyl groups but not by their acyl counterparts. These findings were applied in reactivity-based one-pot assembly of linker-attached Lc(4) and IV(2)Fuc-Lc(4), which are components of human embryonic stem cell surface. The sugar-galectin-1 binding was also examined.

Integrating complex host-pathogen immune environments into S. aureus vaccine studies.

Staphylococcus aureus (SA) is a leading cause of bacterial infection and antibiotic resistance globally. Therefore, development of an effective vaccine has been a major goal of the SA field for the past decades. With the wealth of understanding of pathogenesis, the failure of all SA vaccine trials has been a surprise. We argue that experimental SA vaccines have not worked because vaccines have been studied in naive laboratory animals, whereas clinical vaccine efficacy is tested in immune environments reprogrammed by SA. Here, we review the failed SA vaccines that have seemingly defied all principles of vaccinology. We describe major SA evasion strategies and suggest that they reshape the immune environment in a way that makes vaccines prone to failures. We propose that appropriate integration of concepts of host-pathogen interaction into vaccine study designs could lead to insight critical for the development of an effective SA vaccine.

Non-protective immune imprint underlies failure of Staphylococcus aureus IsdB vaccine.

Humans frequently encounter Staphylococcus aureus (SA) throughout life. Animal studies have yielded SA candidate vaccines, yet all human SA vaccine trials have failed. One notable vaccine "failure" targeted IsdB, critical for host iron acquisition. We explored a fundamental difference between humans and laboratory animals-natural SA exposure. Recapitulating the failed phase III IsdB vaccine trial, mice previously infected with SA do not mount protective antibody responses to vaccination, unlike naive animals. Non-protective antibodies exhibit increased α2,3 sialylation that blunts opsonophagocytosis and preferentially targets a non-protective IsdB domain. IsdB vaccination of SA-infected mice recalls non-neutralizing humoral responses, further reducing vaccine efficacy through direct antibody competition. IsdB vaccine interference was overcome by immunization against the IsdB heme-binding domain. Purified human IsdB-specific antibodies also blunt IsdB passive immunization, and additional SA vaccines are susceptible to SA pre-exposure. Thus, failed anti-SA immunization trials could be explained by non-protective imprint from prior host-SA interaction.

Galectin-1 promotes immunoglobulin production during plasma cell differentiation.

Galectin-1, a beta-galactoside-binding soluble lectin, has been implicated in regulating immune system homeostasis. We investigated the function of galectin-1 in plasma cell differentiation and found that it is induced in primary murine and human differentiating B cells. B lymphocyte-induced maturation protein-1 (Blimp-1), a master regulator for plasma cell differentiation, was necessary and sufficient to induce galectin-1 expression. Notably, ectopic expression of galectin-1 in mature B cells increased Ig mu-chain transcript levels as well as the overall level of Ig production. This function of galectin-1 was dependent on binding to cell surface glycosylated counter receptors, as a galectin-1 mutant deficient in beta-galactoside binding showed diminished ability to promote Ig production. Extracellular galectin-1 bound more significantly to mature B cells than to plasma cells. Lastly, we found that the sugar compound N-acetyllactosamine blocked the binding of galectin-1 to murine splenic B cells and inhibited their differentiation. Taken together, these data are the first to demonstrate a role for galectin-1 in promoting Ig production during plasma cell differentiation.