Molecular characterization of Streptococcus pyogenes (StrepA) non-invasive isolates during the 2022-2023 UK upsurge.

At the end of 2022 into early 2023, the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome-sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016 to 2017. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-2023 were emm1 (28.7 %), emm12 (24.9 %) and emm22 (7.7 %) in throat and emm1 (22 %), emm12 (10 %), emm76 (18 %) and emm49 (7 %) in skin. While all emm1 isolates were the M1UK lineage, the comparison with 2016-2017 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. The analysis of the capsule locus predicted that only 51 % of throat isolates would produce capsule compared with 78% of skin isolates. Ninety per cent of throat isolates were also predicted to have high NADase and streptolysin O (SLO) expression, based on the promoter sequence, compared with only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterize tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges.

Helicobacter pylori promotes gastric cancer through CagA-mediated mitochondrial cholesterol accumulation by targeting CYP11A1 redistribution.

Cholesterol and Helicobacter pylori (H. pylori) are both risk factors for gastric cancer (GC). However, the relationship between cholesterol and H. pylori and their function in the progression of GC are controversial. In this study, we addressed that H. pylori could induce mitochondrial cholesterol accumulation and promote GC proliferation and protect GC cells against apoptosis via cholesterol. Metabolomic and transcriptomic sequencing were used to identify CYP11A1 responsible for H. pylori-induced cholesterol accumulation. In vitro and in vivo function experiments revealed that cholesterol could promote the proliferation of GC and inhibit apoptosis. Mechanically, the interaction of Cytotoxin-associated gene A (CagA) and CYP11A1 redistributed mitochondrial CYP11A1 outside the mitochondria and subsequently caused mitochondrial cholesterol accumulation. The CYP11A1-knockdown upregulated cholesterol accumulation and reproduced the effect of cholesterol on GC in a cholesterol-dependent manner. Moreover, CYP11A1-knockdown or H. pylori infection inhibited mitophagy and maintained the mitochondria homeostasis. H. pylori could contribute to the progression of GC through the CagA/CYP11A1-mitoCHO axis. This study demonstrates that H. pylori can contribute to the progression of GC via cholesterol, and eradicating H. pylori is still prognostically beneficial to GC patients.

On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice.

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

Correlation between the Colony Phenotype and Amino Acid Sequence of the Variable Vaa Antigen in Clinical Isolates of Mycoplasma hominis.

A new Mycoplasma hominis phenotype forming mini-colonies (MC) on agar and distinct from the phenotype forming typical colonies (TC) not only in size, but also in morphology, growth rate, and resistance to adverse factors, has been previously identified. In this study, the phenotype of colonies was determined and a comparative analysis of the amino acid sequence of the main variable antigen Vaa of the laboratory strain N-34 and seven clinical isolates of M. hominis was performed. It is demonstrated that the amino acid sequence of Vaa in clinical isolates forming TC (similar to the laboratory strain N-34) is entirely analogous to that of laboratory strain. Clinical isolates forming MC carry amino acid substitutions in the variable C-terminal region of Vaa, which can contribute to adhesion to eukaryotic cells and immune evasion. The connection between colony phenotype and amino acid sequence of Vaa is established.

Prevalence of the M1UK sublineage among emm1 Streptococcus pyogenes invasive strains isolated in the Czech Republic from December 2022 to May 2023.

AIMS: Since December 2022, an increase in invasive disease caused by Streptococcus pyogenes has been observed in the Czech Republic, with a shift in the clinical presentation and age of patients. Unlike in previous years, invasive disease is more common in children and adolescents under 18 years of age and in previously healthy middle-aged adults. An increase has been noticed in the number of S. pyogenes isolates from primarily sterile sites such as haemoculture, cerebrospinal fluid, pleural effusion fluid, joint fluid, and postmortem specimens. Routine emm gene typing revealed emm1 to be the predominant emm type of S. pyogenes. Between January 2023 and July 2023, 46% of all S. pyogenes isolates from invasive cases were assigned to the emm1 type. The globally spread M1UK sublineage is characterized by differences in the expression of seven genes, including the streptococcal pyrogenic toxin A (speA) gene, compared to historical emm1 iGAS strains. The aim of this study is to determine whether the more toxigenic M1UK sublineage is associated with the increase in invasive disease in the Czech Republic. METHODS: Whole genome sequencing of 41 S. pyogenes isolates from patients with invasive disease recovered in the Czech Republic in 2018 and 2019 and from December 2022 to May 2023 was performed using the MiSeq instrument (Illumina). Bioinformatics analysis was performed using freely available online tools the Bacterial and Viral Bioinformatics Resource Center. RESULTS: Based on whole genome sequencing data of 41 emm1 isolates of S. pyogenes from patients with invasive infectious disease recovered in 2018 and 2019 and from December 2022 to May 2023, the M1UK sublineage was found to be predominant from December 2022 to May 2023. CONCLUSION: The reason for the spread of the M1UK sublineage in the Czech Republic late in 2022 and in the first half of 2023 is not entirely clear, but it may be related to reduced immunity due to limited GAS transmission during lockdowns, especially in children. Another factor that may have contributed to the high incidence of invasive infectious diseases is the seasonal circulation of respiratory viruses.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Helicobacter pylori East Asian type CagA hijacks more SHIP2 by its EPIYA-D motif to potentiate the oncogenicity.

CagA is a significant oncogenic factor injected into host cells by Helicobacter pylori, which is divided into two subtypes: East Asian type (CagAE), characterized by the EPIYA-D motif, and western type (CagAW), harboring the EPIYA-C motif. CagAE has been reported to have higher carcinogenicity than CagAW, although the underlying reason is not fully understood. SHIP2 is an intracellular phosphatase that can be recruited by CagA to perturb the homeostasis of intracellular signaling pathways. In this study, we found that SHIP2 contributes to the higher oncogenicity of CagAE. Co-Immunoprecipitation and Pull-down assays showed that CagAE bind more SHIP2 than CagAW. Immunofluorescence staining showed that a higher amount of SHIP2 recruited by CagAE to the plasma membrane catalyzes the conversion of PI(3,4,5)P3 into PI(3,4)P2. This alteration causes higher activation of Akt signaling, which results in enhanced IL-8 secretion, migration, and invasion of the infected cells. SPR analysis showed that this stronger interaction between CagAE and SHIP2 stems from the higher affinity between the EPIYA-D motif of CagAE and the SH2 domain of SHIP2. Structural analysis revealed the crucial role of the Phe residue at the Y + 5 position in EPIYA-D. After mutating Phe of CagAE into Asp (the corresponding residue in the EPIYA-C motif) or Ala, the activation of downstream Akt signaling was reduced and the malignant transformation of infected cells was alleviated. These findings revealed that CagAE hijacks SHIP2 through its EPIYA-D motif to enhance its carcinogenicity, which provides a better understanding of the higher oncogenic risk of H. pylori CagAE.

Divergent effects of emm types 1 and 12 on invasive group A streptococcal infections-results of a retrospective cohort study, Germany 2023.

As a potential side effect of the severe acute respiratory syndrome coronavirus type 2 pandemic, invasive group A Streptococcus (iGAS) infections in Europe have increased dramatically in both children and adults in the end of 2022. This epidemiological and molecular study describes the distributions of streptococcal genes encoding the M antigen (emm types) and superantigens in patients with invasive and non-invasive GAS infections. From December 2022 to December 2023, a total of 163 GAS isolates were collected from sterile and non-sterile sites of patients at five hospitals in Germany including two tertiary care centers. Genes encoding M protein and superantigens were determined following the guidelines of CDC Streptococcus laboratory. Patients' characteristics were reviewed retrospectively. Correlations of clinical factors, emm types, and superantigens with rates of invasive infections were analyzed. Of the 163 included GAS cases, 112 (69%) were considered as invasive. In total, 33 different emm types were observed, of which emm1.0 (n = 49; 30%), emm89.0 (n = 15; 9%), and emm12.0 (n = 14; 9%) were most prevalent. In total, 70% of emm1.0 isolates belonged to M1UK lineage. No difference in invasive infections was observed for the M1UK lineage compared with other emm1.0 isolates. However, the emm1.0 type, presence of speA1-3, speG, or speJ, as well as adulthood were significantly associated with invasive infections. In contrast, emm12.0 isolates were significantly less associated with invasive infections. Multivariable analysis confirmed a significant influence of speJ and adulthood on iGAS infections. This study underlines the importance of continuous monitoring of genomic trends and identification of emerging GAS variants. This may aid in delineating pathogenicity factors of Streptococcus pyogenes that propel invasive infections.

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.

Exploring the role of E. faecalis enterococcal polysaccharide antigen (EPA) and lipoproteins in evasion of phagocytosis.

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the enterococcal polysaccharide antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called 'decorations', essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

The Helicobacter pylori infection alters the intercellular junctions on the pancreas of gerbils (Meriones unguiculatus).

Helicobacter pylori is a common resident in the stomach of at least half of the world's population and recent evidence suggest its emergence in other organs such as the pancreas. In this organ, the presence of H. pylori DNA has been reported in cats, although the functional implications remain unknown. In this work, we determined distinct features related to the H. pylori manifestation in pancreas in a rodent model, in order to analyse its functional and structural effect. Gerbils inoculated with H. pylori exhibited the presence of this bacterium, as revealed by the expression of some virulence factors, as CagA and OMPs in stomach and pancreas, and confirmed by urease activity, bacterial culture, PCR and immunofluorescence assays. Non-apparent morphological changes were observed in pancreatic tissue of infected animals; however, delocalization of intercellular junction proteins (claudin-1, claudin-4, occludin, ZO-1, E-cadherin, ß-catenin, desmoglein-2 and desmoplakin I/II) and rearrangement of the actin-cytoskeleton were exhibited. This structural damage was consistent with alterations in the distribution of insulin and glucagon, and a systemic inflammation, event demonstrated by elevated IL-8 levels. Overall, these findings indicate that H. pylori can reach the pancreas, possibly affecting its function and contributing to the development of pancreatic diseases.

Geographical distribution, disease association and diversity of Klebsiella pneumoniae K/L and O antigens in India: roadmap for vaccine development.

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K. pneumoniae and its association with geographical distribution, disease severity and antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the Illumina platform, and genomic analysis was carried out using the Kleborate tool. The analysis revealed a total of 78 different KL types, among which KL64 (n=274/1072, 26 %), KL51 (n=249/1072, 24 %), and KL2 (n=88/1072, 8 %) were the most prevalent. In contrast, only 13 distinct O types were identified, with O1/O2v1 (n=471/1072, 44 %), O1/O2v2 (n=353/1072, 33 %), and OL101 (n=66/1072, 6 %) being the predominant serotypes. The study identified 114 different sequence types (STs) with varying serotypes, with ST231 being the most predominant. O serotypes were strongly linked with STs, with O1/O2v1 predominantly associated with ST231. Simpson's diversity index and Fisher's exact test revealed higher serotype diversity in the north and east regions, along with intriguing associations between specific serotypes and resistance profiles. No significant association between KL or O types and disease severity was observed. Furthermore, we found the specific association of virulence factors yersiniabactin and aerobactin (P<0.05) with KL types but no association with O antigen types (P>0.05). Conventionally described hypervirulent clones (i.e. KL1 and KL2) in India lacked typical virulent markers (i.e. aerobactin), contrasting with other regional serotypes (KL51). The cumulative distribution of KL and O serotypes suggests that future vaccines may have to include either ~20 KL or four O types to cover >85 % of the carbapenemase-producing Indian K. pneumoniae population. The results highlight the necessity for comprehensive strategies to manage the diverse landscape of K. pneumoniae strains across different regions in India. Understanding regional serotype dynamics is pivotal for targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse landscape of K. pneumoniae infections across India. This article contains data hosted by Microreact.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

AUF1-mediated inhibition of autophagic lysosomal degradation contributes to CagA stability and Helicobacter pylori-induced inflammation.

CagA, a virulence factor of Helicobacter pylori (H. pylori), is known to drive inflammation in gastric epithelial cells and is typically degraded through autophagy. However, the molecular mechanism by which CagA evades autophagy-mediated degradation remains elusive. This study found that H. pylori inhibits autophagic flux by upregulating the expression of AU-rich element RNA-binding factor 1 (AUF1). We confirmed that AUF1 does not affect autophagy initiation but instead hampers lysosomal clearance, as evidenced by treatments with 3-MA, CQ and BafA1. Upregulated AUF1 stabilizes CagA protein levels by inhibiting the autolysosomal degradation of intracellular CagA in H. pylori-infected gastric epithelial cells. Knocking down AUF1 promotes CagA degradation, an effect that can be reversed by the lysosome inhibitor BafA1 and CQ. Transcriptome analysis of AUF1-knockdown gastric epithelial cells infected with H. pylori indicated that AUF1 regulates the expression of lysosomal-associated hydrolase genes, specifically CTSD, to inhibit autolysosomal degradation. Moreover, we observed that knockdown of AUF1 enhanced the stability of CTSD mRNA and identified AUF1 binding to the 3'UTR region of CTSD mRNA. AUF1-mediated downregulation of CTSD expression contributes to CagA stability, and AUF1 overexpression leads to an increase in CagA levels in exosomes, thus promoting extracellular inflammation. In clinical gastric mucosa, the expression of AUF1 and its cytoplasmic translocation are associated with H. pylori-associated gastritis, with CagA being necessary for the translocation of AUF1 into the cytoplasm. Our findings suggest that AUF1 is a novel host-positive regulator of CagA, and dysregulation of AUF1 expression increases the risk of H. pylori-associated gastritis.

Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Helicobacter pylori CagA Promotes the Formation of Gallstones by Increasing the Permeability of Gallbladder Epithelial Cells.

BACKGROUND: The formation of gallstones is often accompanied by chronic inflammation, and the mechanisms underlying inflammation and stone formation are not fully understood. Our aim is to utilize single-cell transcriptomics, bulk transcriptomics, and microbiome data to explore key pathogenic bacteria that may contribute to chronic inflammation and gallstone formation, as well as their associated mechanisms. METHODS: scRNA-seq data from a gallstone mouse model were extracted from the Gene Expression Omnibus (GEO) database and analyzed using the FindCluster() package for cell clustering analysis. Bulk transcriptomics data from patients with gallstone were also extracted from the GEO database, and intergroup functional differences were assessed using GO and KEGG enrichment analysis. Additionally, 16S rRNA sequencing was performed on gallbladder mucosal samples from asymptomatic patients with gallstone (n = 6) and liver transplant donor gallbladder mucosal samples (n = 6) to identify key bacteria associated with stone formation and chronic inflammation. Animal models were constructed to investigate the mechanisms by which these key pathogenic bacterial genera promote gallstone formation. RESULTS: Analysis of scRNA-seq data from the gallstone mouse model (GSE179524) revealed seven distinct cell clusters, with a significant increase in neutrophil numbers in the gallstone group. Analysis of bulk transcriptomics data from patients with gallstone (GSE202479) identified chronic inflammation in the gallbladder, potentially associated with dysbiosis of the gallbladder microbiota. 16S rRNA sequencing identified Helicobacter pylori as a key bacterium associated with gallbladder chronic inflammation and stone formation. CONCLUSIONS: Dysbiosis of the gallbladder mucosal microbiota is implicated in gallstone disease and leads to chronic inflammation. This study identified H. pylori as a potential key mucosal resident bacterium contributing to gallstone formation and discovered its key pathogenic factor CagA, which causes damage to the gallbladder mucosal barrier. These findings provide important clues for the prevention and treatment of gallstones.

Immunoinformatics-Based Designing of Novel and Potent Multi-Epitope PSA D15 and Cag11 Immunogens for Helicobacter pylori Immunodiagnostic Assay Development.

Helicobacter pylori (H. pylori) strain is the most genetically diverse pathogenic bacterium and now alarming serious human health concern ranging from chronic gastritis to gastric cancer and human death all over the world. Currently, the majority of commercially available diagnostic assays for H. pylori is a challenging task due to the heterogeneity of virulence factors in various geographical regions. In this concern, designing of universal multi-epitope immunogenic biomarker targeted for all H. pylori strains would be crucial to successfully immunodiagnosis assay and vaccine development for H. pylori infection. Hence, the present study aimed to explore the potential immunogenic epitopes of PSA D15 and Cag11 proteins of H. pylori, using immunoinformatics web tools in order to design novel immune-reactive multi-epitope antigens for enhanced immunodiagnosis in humans. Through an in silico immunoinformatics approach, high-ranked B-cell, MHC-I, and MHC-II epitopes of PSA D15 and Cag11 proteins were predicted, screened, and selected. Subsequently, a novel multi-epitope PSA D15 and Cag11 antigens were designed by fused the high-ranked B-cell, MHC-I, and MHC-II epitopes and 50S ribosomal protein L7/L12 adjuvant using linkers. The antigenicity, solubility, physicochemical properties, secondary and tertiary structures, 3D model refinement, and validations were carried. Furthermore, the designed multi-epitope antigens were subjected to codon adaptation and in silico cloning, immune response simulation, and molecular docking with receptor molecules. A novel, stable multi-epitope PSA D15 and Cag11 H. pylori antigens were developed and immune simulation of the designed antigens showed desirable levels of immunological response. Molecular docking of designed antigens with immune receptors (B-cell, MHC-I, MHC-II, and TLR-2/4) revealed robust interactions and stable binding affinity to the receptors. The codon optimized and in silico cloned showed that the designed antigens were successfully expressed (CAI value of 0.95 for PSA D15 and 1.0 for Cag11) after inserted into pET-32ba (+) plasmid of the E. coli K12 strain. In conclusion, this study revealed that the designed multi-epitope antigens have a huge immunological potential candidate biomarker and useful in developing immunodiagnostic assays and vaccines for H. pylori infection.

In silico design and analysis of a multiepitope vaccine against Chlamydia.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG + IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.