Salmonella enterica serovar Typhimurium remodels mitochondrial dynamics of macrophages via the T3SS effector SipA to promote intracellular proliferation.

Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells' mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.

Modulation of MRSA virulence gene expression by the wall teichoic acid enzyme TarO.

Phenol-soluble modulins (PSMs) and Staphylococcal protein A (SpA) are key virulence determinants for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), an important human pathogen that causes a wide range of diseases. Here, using chemical and genetic approaches, we show that inhibition of TarO, the first enzyme in the wall teichoic acid (WTA) biosynthetic pathway, decreases the expression of genes encoding PSMs and SpA in the prototypical CA-MRSA strain USA300 LAC. Mechanistically, these effects are linked to the activation of VraRS two-component system that directly represses the expression of accessory gene regulator (agr) locus and spa. The activation of VraRS was due in part to the loss of the functional integrity of penicillin-binding protein 2 (PBP2) in a PBP2a-dependent manner. TarO inhibition can also activate VraRS in a manner independent of PBP2a. We provide multiple lines of evidence that accumulation of lipid-linked peptidoglycan precursors is a trigger for the activation of VraRS. In sum, our results reveal that WTA biosynthesis plays an important role in the regulation of virulence gene expression in CA-MRSA, underlining TarO as an attractive target for anti-virulence therapy. Our data also suggest that acquisition of PBP2a-encoding mecA gene can impart an additional regulatory layer for the modulation of key signaling pathways in S. aureus.

Characterization of DNA Processing Protein A (DprA) of the Radiation-Resistant Bacterium Deinococcus radiodurans.

Environmental DNA uptake by certain bacteria and its integration into their genome create genetic diversity and new phenotypes. DNA processing protein A (DprA) is part of a multiprotein complex and facilitates the natural transformation (NT) phenotype in most bacteria. Deinococcus radiodurans, an extremely radioresistant bacterium, is efficient in NT, and its genome encodes nearly all of the components of the natural competence complex. Here, we have characterized the DprA protein of this bacterium (DrDprA) for the known characteristics of DprA proteins of other bacteria and the mechanisms underlying the DNA-RecA interaction. DrDprA has three domains. In vitro studies showed that purified recombinant DrDprA binds to both single-strand DNA (ssDNA) and double-strand DNA (dsDNA) and is able to protect ssDNA from nucleolytic degradation. DrDprA showed a strong interaction with DrRecA and facilitated RecA-catalyzed functions in vivo. Mutational studies identified DrDprA amino acid residues crucial for oligomerization, the interaction with DrRecA, and DNA binding. Furthermore, we showed that both oligomerization and DNA binding properties of DrDprA are integral to its support of the DrRecA-catalyzed strand exchange reaction (SER) in vitro. Together, these data suggested that DrDprA is largely structurally conserved with other DprA homologs but shows some unique structure-function features like the existence of an additional C-terminal Drosophila melanogaster Miasto-like protein 1 (DML1) domain, equal affinities for ssDNA and dsDNA, and the collective roles of oligomerization and DNA binding properties in supporting DrRecA functions. IMPORTANCE Bacteria can take up extracellular DNA (eDNA) by natural transformation (NT). Many bacteria, including Deinococcus radiodurans, have constitutive competence systems and can take up eDNA throughout their growth phase. DprA (DNA processing protein A) is a transformation-specific recombination mediator protein (RMP) that plays a role in bacterial NT, and the absence of this gene significantly reduces the transformation efficiencies of both chromosomal and plasmid DNA. NT helps bacteria survive under adverse conditions and contributes to genetic diversity in bacteria. The present work describes the characterization of DprA from D. radiodurans and will add to the existing knowledge of DprA biology.

The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response.

Streptococcus agalactiae is a leading cause of infections in neonates. This opportunistic pathogen colonizes the vagina, where it has to cope with acidic pH and hydrogen peroxide produced by lactobacilli. Thus, in the host, this bacterium possesses numerous adaptation mechanisms in which the pleiotropic regulators play a major role. The transcriptional regulator CcpA (catabolite control protein A) has previously been shown to be the major regulator involved in carbon catabolite repression in Gram-positive bacteria but is also involved in other functions. By transcriptomic analysis, we characterized the CcpA-dependent gene regulation in S. agalactiae. Approximately 13.5% of the genome of S. agalactiae depends on CcpA for regulation and comprises genes involved in sugar uptake and fermentation, confirming the role of CcpA in carbon metabolism. We confirmed by electrophoretic mobility shift assays (EMSAs) that the DNA binding site called cis-acting catabolite responsive element (cre) determined for other streptococci was effective in S. agalactiae. We also showed that CcpA is of capital importance for survival under acidic and oxidative stresses and is implicated in macrophage survival by regulating several genes putatively or already described as involved in stress response. Among them, we focused our study on SAK_1689, which codes a putative UspA protein. We demonstrated that SAK_1689, highly downregulated by CcpA, is overexpressed under oxidative stress conditions, this overexpression being harmful for the bacterium in a ΔccpA mutant. IMPORTANCE Streptococcus agalactiae is a major cause of disease burden leading to morbidity and mortality in neonates worldwide. Deciphering its adaptation mechanisms is essential to understand how this bacterium manages to colonize its host. Here, we determined the regulon of the pleiotropic regulator CcpA in S. agalactiae. Our findings reveal that CcpA is not only involved in carbon catabolite repression, but is also important for acidic and oxidative stress resistance and survival in macrophages.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells.

BACKGROUND: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1ß and TNF-α cytokines was measured by ELISA. RESULTS: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-ß as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). CONCLUSION: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Characterization of SCCmec Instability in Methicillin-Resistant Staphylococcus aureus Affecting Adjacent Chromosomal Regions, Including the Gene for Staphylococcal Protein A (spa).

Staphylococcal cassette chromosome mec (SCCmec) represents a sequence of clear clinical and diagnostic importance in staphylococci. At a minimum the chromosomal cassette contains the mecA gene encoding PBP2a but frequently also includes additional antibiotic resistance genes (e.g., ermA and aadC; macrolide and aminoglycoside resistance, respectively). Certain regions within SCCmec elements are hot spots for sequence instability due to cassette-specific recombinases and a variety of internal mobile elements. SCCmec changes may affect not only cassette stability but the integrity of adjacent chromosomal sequences (e.g., the staphylococcal protein A gene; spa). We investigated SCCmec stability in methicillin-resistant Staphylococcus aureus (MRSA) strains carrying one of four SCCmec types cultured in the absence of antimicrobial selection over a 3-month period. SCCmec rearrangements were first detected in cefoxitin-susceptible variants after 2 months of passage, and most commonly showed precise excision of the SCCmec element. Sequence analysis after 3 months revealed both precise SCCmec excision and a variety of SCCmec internal deletions, some including extensive adjacent chromosomal loss, including spa. No empty cassettes (i.e., loss of just mecA from SCCmec) were observed among the variants. SCCmec stability was influenced both by internal mobile elements (IS431) as well as the host cell environment. Genotypically similar clinical isolates with deletions in the spa gene were also included for purposes of comparison. The results indicate a role for host-cell influence and the IS431 element on SCCmec stability.

Genome-Wide Mapping and Microscopy Visualization of Protein-DNA Interactions by pA-DamID.

Several methods have been developed to map protein-DNA interactions genome-wide in the last decades. Protein A-DamID (pA-DamID) is a recent addition to this list with distinct advantages. pA-DamID relies on antibody-based targeting of the bacterial Dam enzyme, resulting in adenine methylation of DNA in contact with the protein of interest. This m6A can then be visualized by microscopy, or mapped genome-wide. The main advantages of pA-DamID are an easy and direct visualization of DNA that is in contact with the protein of interest, unbiased mapping of protein-DNA interactions, and the possibility to select specific subpopulations of cells by flow cytometry before further sample processing. pA-DamID is particularly suited to study proteins that form large chromatin domains or that are part of distinct nuclear structures such as the nuclear lamina. This chapter describes the pA-DamID procedure from cell harvesting to the preparation of microscopy slides and high-throughput sequencing libraries.

Molecular epidemiology and phenotypes of invasive methicillin-resistant vancomycin-intermediate Staphylococcus aureus in Taiwan.

BACKGROUND: Patients with invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA), especially those with an elevated minimal inhibitory concentration (MIC) of vancomycin (VA), are likely to have treatment failure and poor outcomes. The aim of this study was to delineate and correlate the genotypes and phenotypes of clinical VA-intermediate S. aureus (VISA) from invasive infections in Taiwan. METHODS: Between 2006 and 2010, a total of 670 non-duplicate MRSA isolates were collected from patients with invasive infections, mostly from blood, as part of a nationwide antimicrobial surveillance program named Tigecycline in vitro Surveillance in Taiwan. Among them, 10 (1.5%) VISA (VA MIC = 4 mg/L) isolates were identified. Molecular typing with staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing, staphylococcal protein A (spa), mec-associated hypervariable region (dru), accessory gene regulator (agr), and pulse-field gel electrophoresis, and phenotypic analysis including antibiotic susceptibility testing, gene encoding Panton-Valentine leukocidin (pvl), and superantigenic toxin profiles, were analyzed. RESULTS: All but one isolate was defined as molecular health-care-associated MRSA: 6 as SCCmecIII-ST239-spa t037-agrI-dru7 (1 isolate) and dru14 (5 isolates), 2 as SCCmecII-ST5-spa t586-agrII-dru4, and one as SCCmecII-ST89-spa t3520-agrIII-dru7. One isolate was defined as SCCmecIV-ST59-spa t437-agrI-dru8, which was categorized as molecular community-associated MRSA. Five pulsotypes were identified; only one had a positive D-test and 3 were insusceptible to daptomycin (MIC ≧1 mg/L). Five isolates possessed sea-selk-selq, among them 4 belonged to SCCmecIII-ST239-spa t037-agrI. CONCLUSION: In this study, VISA was rarely isolated from invasive MRSA infections, and most cases harbored limited genotypes and corresponding phenotypes.

Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag.

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

Global diversity of the gene encoding the Pfs25 protein-a Plasmodium falciparum transmission-blocking vaccine candidate.

BACKGROUND: Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations. METHODS: A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures. RESULTS: The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection. CONCLUSIONS: The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control.

Clonality, virulence genes, and antibiotic resistance of Staphylococcus aureus isolated from blood in Shandong, China.

BACKGROUND: Bloodstream infection (BSI) caused by Staphylococcus aureus (S. aureus) can be life-threatening and pose a great challenge to infection control and clinical treatment. However, little information exists regarding the characterization of S. aureus in BSI patients in Shandong, China. To identify the clonality, virulence genes, and antibiotic resistance of S. aureus in blood, a total of 101 nonrepetitive blood isolates were collected. The antibiotic resistance phenotypes were determined, and virulence genes were analyzed with polymerase chain reaction (PCR). Finally, the genetic relatedness was investigated with Staphylococcus chromosomal cassette mec (SCCmec) typing for methicillin-resistant S. aureus (MRSA) isolates, Staphylococcal protein A (spa), and multilocus sequence typing (MLST) for all of 101 isolates. RESULTS: Of the 101 S. aureus isolates, 24 MRSA isolates and 77 methicillin-susceptible S. aureus (MSSA) isolates were identified. Overall, MRSA isolates had higher resistance rates than MSSA isolates when exposed to any of the 15 antibiotics tested in this study except for trimethoprim/sulfamethoxazole. Among the 17 virulence genes tested in this study, hla, hld, and hlg could be detected in all isolates. MRSA isolates were more likely to carry seb and hlb genes, while MSSA isolates were more likely to carry seg and sei genes. Thirty-five sequence types (STs) and 49 spa types were identified, of which ST59-t437 and ST398-t571 were the most abundant. These two genotypes were also the most abundant ST-spa types in MRSA and MSSA isolates, but their abundances shifted over time, with ST398-t571 being the predominant genotype from 2016 to 2017, and ST59-t437 from 2018 to 2020. Besides, all the ST59-t437 isolates harbored hlgb gene, whereas most (88.9%) ST398-t571 did not. In addition, twenty-four MRSA isolates were subject to SCCmec typing. SCCmec IVa was the most prevalent SCCmec type, and all the ST59-t437 MRSA isolates were SCCmec IVa. We also observed 15 new STs, and some of them were MRSA. CONCLUSION: These findings provide additional observations and epidemiological data for blood S. aureus isolates, which can improve future infection-control measures and aid in potential clinical treatments in hospitals and other clinical settings.

Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide.

BACKGROUND: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. RESULTS: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. CONCLUSIONS: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

A protein A based Staphylococcus aureus vaccine with improved safety.

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with Fab encoded by VH3 clan related genes. SpA binding to VH3-IgM that serves as a B cell receptor results in B cell expansion and secretion of antibodies with no specificity for S. aureus. SpA crosslinking of VH3-IgG and VH3-IgE bound to cognate receptors of mast cells and basophils promotes histamine release and anaphylaxis. Earlier work developed a prototype variant SpAKKAA with four amino acid substitutions in each IgBD. When tested in animal models, SpAKKAA elicited neutralizing antibodies and protection against infection. We show here that SpAKKAA retains crosslinking activity for VH3-IgG and VH3-IgE. We use a rational approach to design and test 67 new SpA variants for loss of VH3 binding and anaphylactic activities. We identify two detoxified candidates that elicit SpA-neutralizing antibodies and protect animals from S. aureus colonization and bloodstream infection. The new detoxified SpA candidates bear three instead of four amino acid substitutions thus increasing the development of SpA-specific antibodies. We propose that detoxified SpA variants unable to crosslink VH3-idiotypic immunoglobulin may be suitably developed as clinical-grade vaccines for safety and efficacy testing in humans.

Consideration on Efficient Recombinant Protein Production: Focus on Substrate Protein-Specific Compatibility Patterns of Molecular Chaperones.

Expression of recombinant proteins requires at times the aid of molecular chaperones for efficient post-translational folding into functional structure. However, predicting the compatibility of a protein substrate with the right type of chaperone to produce functional proteins is a daunting issue. To study the difference in effects of chaperones on His-tagged recombinant proteins with different characteristics, we performed in vitro proteins expression using Escherichia coli overexpressed with several chaperone 'teams': Trigger Factor (TF), GroEL/GroES and DnaK/DnaJ/GrpE, alone or in combinations, with the aim to determine whether protein secondary structure can serve as predictor for chaperone success. Protein A, which has a helix dominant structure, showed the most efficient folding with GroES/EL or TF chaperones alone, whereas Protein B, which has less helix in the structure, showed a remarkable effect on the DnaK/J/GrpE system alone. This tendency was also seen with other recombinant proteins with particular properties. With the chaperons' assistance, both proteins were synthesized more efficiently in the culture at 22.5 °C for 20 h than at 37 °C for 3 h. These findings suggest a novel avenue to study compatibility of chaperones with substrate proteins and optimal culture conditions for producing functional proteins with a potential for predictive analysis of the success of chaperones based on the properties of the substrate protein.

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A.

Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.IMPORTANCE The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.

Temporal Variation in Staphylococcus aureus Protein A Genotypes from Nose and Skin in Atopic Dermatitis Patients.

BACKGROUND: Staphylococcus aureus colonization is associated with disease severity in patients with atopic dermatitis (AD). OBJECTIVE: To investigate temporal variation in S. aureus protein A gene (spa)-types isolated from the nose and lesional skin and the correlation of spa-types with disease severity. RESULTS: This study included 96 adult AD patients who were assessed at baseline (T0) and after a strict 2-week follow-up period (T1) in which treatment was standardized with a topical corticosteroid. Fifty-five different spa-types were detected in the nose and skin cultures. Seventy-three patients were colonized with S. aureus in the nasal cavity at both time points (persistent carriership), 59 of whom (81%) had identical spa-types over time. For skin samples, 42 (75%) of the 56 persistent skin carriers had identical spa-types over time. The same spa-type was carried in the nose and skin in 79 and 77% of the patients at T0 and T1, respectively. More severe disease was not associated with specific spa-types or with temporal variation in spa-type. CONCLUSION: S. aureus strains in AD are highly heterogeneous between patients. The majority of patients carry the same spa-type in the nose and skin without temporal variation, suggesting clonal colonization within individual patients. No predominant spa-type or temporal variation is associated with increased disease severity.

Protein A of Staphylococcus aureus strain NCTC8325 interacted with heparin.

Heparin, known for its anticoagulant activity, is commonly used as the coatings of medical devices. The attaching of Staphylococcus aureus, a prominent human and animal pathogen, to the heparin coatings usually leads to catheter-related bloodstream infections. Hence, the study of the interaction between heparin and S. aureus surface proteins is desired. Here, we found that protein A (SpA) of S. aureus was a heparin-binding protein, contributing to the interaction between S. aureus and heparin. The cell-wall-anchored SpA was one of the most critical S. aureus virulence factors with a lysin-like motif (LysM). When SpA was mutated to remove the LysM motif, the heparin-binding capability of SpA dropped 50%. The in-frame deletion of spa also reduced the heparin-binding capability of S. aureus. There was 1.3-fold more of heparin bound to wild type S. aureus than the Δspa::Em strain. These results would help understand the host-microbe interaction and the infection by S. aureus.

Nonsteroidal antiinflammatory drugs alter antibiotic susceptibility and expression of virulence-related genes and protein A of Staphylococcus aureus

Background/aim: Nonsteroidal antiinflammatory drugs (NSAIDs) including diclofenac, naproxen, ibuprofen, acetylsalicylic acid, and acetaminophen have been shown to have antimicrobial effects on various microorganisms. The aim of this study was to investigate the antibacterial effects of NSAIDs on Staphylococcus aureus. Materials and methods: Susceptibilities of S. aureus strains to NSAIDs with or without antimicrobials (moxifloxacin, vancomycin, ciprofloxacin, clindamycin, and gentamicin) were determined using the microdilution method and disk diffusion test. Expression levels of genes in the presence of drugs were investigated by real-time quantitative RT-PCR (qRT-PCR), and immunoblotting analysis was performed for staphylococcal protein A (SpA). Results: Our results showed that all NSAIDs were active against S. aureus strains with MIC values ranging from 195 µg/mL to 6250 µg/ mL. NSAIDs increased the antibiotic susceptibility of the strains, and diclofenac was found to be more effective than the other drugs. Drugs showed different effects on expression levels of virulence factor and/or regulatory genes. Immunoblotting analysis of SpA protein was mostly in accordance with qRT-PCR results. Conclusion: The regulatory/virulence factor genes and proteins of S. aureus investigated in this study may be reasonable targets for these drugs, and we suggest that the data may contribute to the field of infection control and antimicrobial resistance.

Simultaneously targeting nitrocellulose and antibody by a dual-headed protein.

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix - a protein that has a strong affinity to nitrocellulose membrane and protein A - a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.