Search

Abstract Isogenic bacterial cell populations are phenotypically heterogenous and may include subpopulations of antibiotic tolerant or heteroresistant cells. The reversibility of these phenotypes and lack of biomarkers to differentiate functionally different, but morphologically identical cells is a challenge for research and clinical detection. To overcome this, we present ´Cellular Phenotypic Profiling and backTracing (CPPT)´, a fluorescence-activated cell sorting platform that uses fluorescent probes to visualize and quantify cellular traits and connects this phenotypic profile with a cell´s experimentally determined fate in single cell-derived growth and antibiotic susceptibility analysis. By applying CPPT on Staphylococcus aureus we phenotypically characterized dormant cells, exposed bimodal growth patterns in colony-derived cells and revealed different culturability of single cells on solid compared to liquid media. We demonstrate that a fluorescent vancomycin conjugate marks cellular subpopulations of vancomycin-intermediate S. aureus with increased likelihood to survive antibiotic exposure, showcasing the value of CPPT for discovery of clinically relevant biomarkers.

ABSTRACT The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus.IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.

ABSTRACTIn the Staphylococcus aureus genome, a set of highly conserved two-component systems (TCSs) composed of histidine kinases (HKs) and their cognate response regulators (RRs) sense and respond to environmental stimuli, which drive the adaptation of the bacteria. This study investigates the complex interplay between TCSs in S. aureus USA300, a predominant methicillin-resistant S. aureus strain, revealing shared and unique virulence regulatory pathways and genetic variations mediating signal specificity within TCSs. Using TCS-related mutants from the Nebraska Transposon Mutant Library, we analyzed the effects of inactivated TCS HKs and RRs on the production of various virulence factors, in vitro infection abilities, and adhesion assays. We found that the TCSs’ influence on virulence determinants was not associated with their phylogenetic relationship, indicating divergent functional evolution. Using the co-crystallized structure of the DesK-DesR from Bacillus subtilis and the modeled structures of the four NarL TCSs in S. aureus, we identified interacting residues, revealing specificity determinants and conservation within the same TCS, even from different strain backgrounds. The interacting residues were highly conserved within strains but varied between species due to selection pressures and the coevolution of cognate pairs. This study unveils the complex interplay and divergent functional evolution of TCSs, highlighting their potential for future experimental exploration of phosphotransfer between cognate and non-cognate recombinant HK and RRs.IMPORTANCEGiven the widespread conservation of two-component systems (TCSs) in bacteria and their pivotal role in regulating metabolic and virulence pathways, they present a compelling target for anti-microbial agents, especially in the face of rising multi-drug-resistant infections. Harnessing TCSs therapeutically necessitates a profound understanding of their evolutionary trajectory in signal transduction, as this underlies their unique or shared virulence regulatory pathways. Such insights are critical for effectively targeting TCS components, ensuring an optimized impact on bacterial virulence, and mitigating the risk of resistance emergence via the evolution of alternative pathways. Our research offers an in-depth exploration of virulence determinants controlled by TCSs in S. aureus, shedding light on the evolving specificity determinants that orchestrate interactions between their cognate pairs.

Abstract Background The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. Results S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. Conclusion We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.

IntroductionImproved understanding of Staphylococcus aureus throat colonization in the presence of other co-existing microbes is important for mapping S. aureus adaptation to the human throat, and recurrence of infection. Here, we explore the responses triggered by the encounter between two common throat bacteria, S. aureus and Streptococcus anginosus, to identify genes in S. aureus that are important for colonization in the presence of human tonsillar epithelial cells and S. anginosus, and further compare this transcriptome with the genes expressed in S. aureus as only bacterium.MethodsWe performed an in vitro co-culture experiment followed by RNA sequencing to identify interaction-induced transcriptional alterations and differentially expressed genes (DEGs), followed by gene enrichment analysis.Results and discussionA total of 332 and 279 significantly differentially expressed genes with p-value < 0.05 and log2 FoldChange (log2FC) ≥ |2| were identified in S. aureus after 1 h and 3 h co-culturing, respectively. Alterations in expression of various S. aureus survival factors were observed when co-cultured with S. anginosus and tonsillar cells. The serine-aspartate repeat-containing protein D (sdrD) involved in adhesion, was for example highly upregulated in S. aureus during co-culturing with S. anginosus compared to S. aureus grown in the absence of S. anginosus, especially at 3 h. Several virulence genes encoding secreted proteins were also highly upregulated only when S. aureus was co-cultured with S. anginosus and tonsillar cells, and iron does not appear to be a limiting factor in this environment. These findings may be useful for the development of interventions against S. aureus throat colonization and could be further investigated to decipher the roles of the identified genes in the host immune response in context of a throat commensal landscape.

Infected chronic skin wounds and other skin infections are increasingly putting pressure on the health care providers and patients. The pressure is especially concerning due to the rise of antimicrobial resistance and biofilm-producing bacteria that further impair treatment success. Therefore, innovative strategies for wound healing and bacterial eradication are urgently needed; utilization of materials with inherent biological properties could offer a potential solution. Chitosan is one of the most frequently used polymers in delivery systems. This bioactive polymer is often regarded as an attractive constituent in delivery systems due to its inherent antimicrobial, anti-inflammatory, anti-oxidative, and wound healing properties. However, lipid-based vesicles and liposomes are generally considered more suitable as delivery systems for skin due to their ability to interact with the skin structure and provide prolonged release, protect the antimicrobial compound, and allow high local concentrations at the infected site. To take advantage of the beneficial attributes of the lipid-based vesicles and chitosan, these components can be combined into chitosan-containing liposomes or chitosomes and chitosan-coated liposomes. These systems have previously been investigated for use in wound therapy; however, their potential in infected wounds is not fully investigated. In this study, we aimed to investigate whether both the chitosan-containing and chitosan-coated liposomes tailored for infected wounds could improve the antimicrobial activity of the membrane-active antimicrobial chlorhexidine, while assuring both the anti-inflammatory activity and cell compatibility. Chlorhexidine was incorporated into three different vesicles, namely plain (chitosan-free), chitosan-containing and chitosan-coated liposomes that were optimized for skin wounds. Their release profile, antimicrobial activities, anti-inflammatory properties, and cell compatibility were assessed in vitro. The vesicles comprising chitosan demonstrated slower release rate of chlorhexidine and high cell compatibility. Additionally, the inflammatory responses in murine macrophages treated with these vesicles were reduced by about 60% compared to non-treated cells. Finally, liposomes containing both chitosan and chlorhexidine demonstrated the strongest antibacterial effect against Staphylococcus aureus. Both chitosan-containing and chitosan-coated liposomes comprising chlorhexidine could serve as excellent platforms for the delivery of membrane-active antimicrobials to infected wounds as confirmed by improved antimicrobial performance of chlorhexidine.

Abstract Aim Delirium is a serious, acute medical condition which places a heavy burden on the patient, his or her family and healthcare professionals. There have been only a limited number of studies to explore nurses' experiences of delirium and how delirium is identified in community care. The research questions of the study are as follows: “How do community care nurses' experience delirium?” and “How is delirium identified?”. Design This study has been designed as an explorative and descriptive study. Methods A topic‐based interview guide was developed containing questions associated with the Registered Nurses' experiences of their meetings with people with delirium and their identification of delirium. Results Nurses working in the community care need to know more about delirium as they play a key role in treatment. Our results also show that the participants have difficulty in establishing whether a patient is suffering from acute confusion/delirium, depression or dementia.

Abstract Background Blood culture bottles (BCBs) provide a semiautomated method for culturing periprosthetic tissue specimens. A study evaluating BCBs for culturing clinical samples other than body fluids is needed before implementation into clinical practice. Our objective was to evaluate use of the BacT/Alert® Virtuo blood culture system for culturing periprosthetic tissue specimens. Methods The study was performed through the analysis of spiked (n = 36) and clinical (n = 158) periprosthetic tissue samples. Clinical samples were analyzed by the BCB method and the results were compared to the conventional microbiological culture-based method for time to detection and microorganisms identified. Results The BacT/Alert® Virtuo blood culture system detected relevant bacteria for prosthetic joint infection in both spiked and clinical samples. The BCB method was found to be as sensitive (79%) as the conventional method (76%) (p = 0.844) during the analyses of clinical samples. The BCB method yielded positive results much faster than the conventional method: 89% against 27% detection within 24 h, respectively. The median detection time was 11.1 h for the BCB method (12 h and 11 h for the aerobic and the anaerobic BCBs, correspondingly). Conclusion We recommend using the BacT/Alert® Virtuo blood culture system for analyzing prosthetic joint tissue, since this detect efficiently and more rapidly a wider range of bacteria than the conventional microbiological method.

Bacterial membrane vesicles (MVs) mediate bacterial virulence by enabling secretion and long distance delivery of bacterial effector molecules. Staphylococcus haemolyticus has now been demonstrated to produce membrane vesicles (MVs). The protein content of S. haemolyticus MVs was identified by Mass spectrometry and compared to proteins identified in the total secretome. This information is presented in this data article. Further background and interpretation of the data can be found in the article: Comparative exoproteome profiling of an invasive and a commensal S. haemolyticus isolate (Cavanagh et al., in press). Data are available via Proteome Xchange with identifier PXD010389.

Bacterial extracellular vesicles (EVs) have a vital role in bacterial pathogenesis. However, to date, the small RNA-cargo of EVs released by the opportunistic pathogen Staphylococcus aureus has not been characterized. Here, we shed light on the association of small RNAs with EVs secreted by S. aureus MSSA476 cultured in iron-depleted bacteriologic media supplemented with a subinhibitory dosage of vancomycin to mimic infection condition. Confocal microscopy analysis on intact RNase-treated EVs indicated that RNA is associated with EV particles. Transcriptomic followed by bioinformatics analysis of EV-associated RNA revealed the presence of potential gene regulatory small RNAs and high levels of tRNAs. Among the EV-associated enriched small RNAs were SsrA, RsaC and RNAIII. Our finding invites new insights into the potential role of EV-associated RNA as a modulator of host-pathogen interaction.

Clinical metagenomics is actively moving from research to clinical laboratories. It has the potential to change the microbial diagnosis of infectious diseases, especially when detection and identification of pathogens can be challenging, such as in prosthetic joint infection (PJI). The application of metagenomic sequencing to periprosthetic joint tissue (PJT) specimens is often challenged by low bacterial load in addition to high level of inhibitor and contaminant host DNA, limiting pathogen recovery. Shotgun-metagenomics (SMg) performed directly on positive blood culture bottles (BCBs) inoculated with PJT may be a convenient approach to overcome these obstacles. The aim was to test if it is possible to perform SMg on PJT inoculated into BCBs for pathogen identification in PJI diagnosis. Our study was conducted as a laboratory method development. For this purpose, spiked samples (positive controls), negative control and clinical tissue samples (positive BCBs) were included to get a comprehensive overview. We developed a method for preparation of bacterial DNA directly from PJT inoculated in BCBs. Samples were processed using MolYsis5 kit for removal of human DNA and DNA extracted with BiOstic kit. High DNA quantity/quality was obtained, and no inhibition was observed during the library preparation, allowing further sequencing process. DNA sequencing reads obtained from the BCBs, presented a low proportion of human reads (

Abstract Background Staphylococcus aureus cell wall anchored Serine Aspartate repeat containing protein D (SdrD) is a member of the microbial surface component recognising adhesive matrix molecules (MSCRAMMs). It is involved in the bacterial adhesion and virulence. However the extent of genetic variation in S. aureus sdrD gene within isolates from healthy carriers are not known. The aim of this study was to evaluate allelic variation of the sdrD gene among S. aureus from healthy nasal carriers. Results The sdrD A region from 48 S. aureus isolates from healthy carriers were analysed and classified into seven variants. Variations in the sdrD A region were concentrated in the N2 and N3 subdomains. Sequence analysis of the entire sdrD gene of representative isolates revealed variations in the SD repeat and the EF motifs of the B repeat. In silico structural modelling indicates that there are no differences in the SdrD structure of the 7 variants. Variable amino acid residues mapped onto the 3D structure revealed that the variations are surface located, exist within the groove between the N2-N3 subdomains and distributed mainly on the N3 subdomain. Comparison of adhesion to keratinocytes in an in vitro cell adhesion assay, using NCTC 8325–4∆sdrD strains expressing the various sdrD gene variants, indicated a significant difference between only two complements while others showed no major difference in their adhesion. Conclusions This study provides evidence of sequence variations across the different domains of SdrD from S. aureus isolated from healthy nasal carriers. Proper understanding of these variations is necessary in the study of S. aureus pathogenesis.

An active wound dressing should address the main goals in wound treatment, which are improved wound healing and reduced infection rates. We developed novel multifunctional nanofibrous wound dressings with three active ingredients: chloramphenicol (CAM), beta-glucan (βG) and chitosan (CHI), of which βG and CHI are active nanofiber-forming biopolymers isolated from the cell walls of Saccharomyces cerevisiae and from shrimp shells, respectively. To evaluate the effect of each active ingredient on the nanofibers’ morphological features and bioactivity, nanofibers with both βG and CHI, only βG, only CHI and only copolymers, polyethylene oxide (PEO) and hydroxypropylmethylcellulose (HPMC) were fabricated. All four nanofiber formulations were also prepared with 1% CAM. The needle-free NanospiderTM technique allowed for the successful production of defect-free nanofibers containing all three active ingredients. The CAM-containing nanofibers had a burst CAM-release and a high absorption capacity. Nanofibers with all active ingredients (βG, CHI and CAM) showed a concentration-dependent anti-inflammatory activity, while maintaining the antimicrobial activity of CAM. The promising anti-inflammatory properties, together with the high absorption capacity and antimicrobial effect, make these multifunctional nanofibers promising as dressings in local treatment of infected and exuding wounds, such as burn wounds.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

Abstract Background Colonization of the body is an important step in Staphylococcus aureus infection. S. aureus colonizes skin and mucous membranes in humans and several animal species. One important ecological niche of S. aureus is the anterior nares. More than 60% of the S. aureus in the nose are found in vestibulum nasi. Our aim was to describe the localization of S. aureus in nasal tissue from healthy carriers. Methods Punch skin biopsies were taken from vestibulum nasi from healthy volunteers (S. aureus carriers and non−/intermittent carriers, n = 39) attending the population-based Tromsø 6 study. The tissue samples were processed as frozen sections before immunostaining with a specific S. aureus antibody, and finally evaluated by a confocal laser-scanning microscope. Results Our results suggest that S. aureus colonize both the upper and lower layers of the epidermis within the nasal epithelium of healthy individuals. The number of S. aureus in epidermis was surprisingly low. Intracellular localization of S. aureus in nasal tissue from healthy individuals was also detected. Conclusions Knowledge of the exact localization of S. aureus in nasal tissue is important for the understanding of the host responses against S. aureus. Our results may have consequences for the eradication strategy of S. aureus in carriers, and further work can provide us with tools for targeted prevention of S. aureus colonisation and infection.

Burns and other skin injuries are growing concerns as well as challenges in an era of antimicrobial resistance. Novel treatment options to improve the prevention and eradication of infectious skin biofilm-producing pathogens, while enhancing wound healing, are urgently needed for the timely treatment of infection-prone injuries. Treatment of acute skin injuries requires tailoring of formulation to assure both proper skin retention and the appropriate release of incorporated antimicrobials. The challenge remains to formulate antimicrobials with low water solubility, which often requires carriers as the primary vehicle, followed by a secondary skin-friendly vehicle. We focused on widely used chlorhexidine formulated in the chitosan-infused nanocarriers, chitosomes, incorporated into chitosan hydrogel for improved treatment of skin injuries. To prove our hypothesis, lipid nanocarriers and chitosan-comprising nanocarriers (≈250 nm) with membrane-active antimicrobial chlorhexidine were optimized and incorporated into chitosan hydrogel. The biological and antibacterial effects of both vesicles and a vesicles-in-hydrogel system were evaluated. The chitosomes-in-chitosan hydrogel formulation demonstrated promising physical properties and were proven safe. Additionally, the chitosan-based systems, both chitosomes and chitosan hydrogel, showed an improved antimicrobial effect against S. aureus and S. epidermidis compared to the formulations without chitosan. The novel formulation could serve as a foundation for infection prevention and bacterial eradication in acute wounds.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infections and causes serious reproductive tract complications among women. The limitations of existing oral antibiotics and treatment of antimicrobial resistance require alternative treatment options. We are proposing, for the first time, the natural polyphenol resveratrol (RES) in an advanced delivery system comprising liposomes incorporated in chitosan hydrogel, for the localized treatment of C. trachomatis infection. Both free RES and RES liposomes-in-hydrogel inhibited the propagation of C. trachomatis in a concentration-dependent manner, assessed by the commonly used in vitro model comprising McCoy cells. However, for lower concentrations, the anti-chlamydial effect of RES was enhanced when incorporated into a liposomes-in-hydrogel delivery system, with inhibition of 78% and 94% for 1.5 and 3 µg/mL RES, respectively for RES liposomes-in-hydrogel, compared to 43% and 72%, respectively, for free RES. Furthermore, RES liposomes-in-hydrogel exhibited strong anti-inflammatory activity in vitro, in a concentration-dependent inhibition of nitric oxide production in the LPS-induced macrophages (RAW 264.7). The combination of a natural substance exhibiting multi-targeted pharmacological properties, and a delivery system that provides enhanced activity as well as applicability for vaginal administration, could be a promising option for the localized treatment of C. trachomatis infection.

Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus-derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo. Finally, immunization of mice with S. aureus-derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial–host interactions during systemic infections and provide protective immunity in murine models of infection.

Enterococcus faecium has undergone a transition to a multidrug-resistant nosocomial pathogen. The population structure of E. faecium is characterized by a sharp distinction of clades, where the hospital-adapted lineage is primarily responsible for bacteremia. So far, factors that were identified in hospital-adapted strains and that promoted pathogenesis of nosocomial E. faecium mainly play a role in adherence and biofilm production, while less is known about factors contributing to survival in blood. This study identified a gene cluster, which includes genes encoding bacterial Toll/interleukin-1 receptor- (TIR-) domain-containing proteins (TirEs). The cluster was found to be unique to nosocomial strains and to be located on a putative mobile genetic element of phage origin. The three genes within the cluster appeared to be expressed as an operon. Expression was detected in bacterial culture media and in the presence of human blood. TirEs are released into the bacterial supernatant, and TirE2 is associated with membrane vesicles. Furthermore, the tirE-gene cluster promotes bacterial proliferation in human blood, indicating that TirE may contribute to the pathogenesis of bacteremia.

Topical administration of drugs into the vagina can provide local therapy of vaginal infections, preventing the possible systemic side effects of the drugs. The natural polysaccharide chitosan is known for its excellent mucoadhesive properties, safety profile, and antibacterial effects, and thus it can be utilized in improving localized vaginal therapy by prolonging the residence time of a drug at the vaginal site while acting as an antimicrobial in synergy. Therefore, we aimed to explore the potential of chitosan, namely chitosan-coated liposomes and chitosan hydrogel, as an excipient with intrinsic antimicrobial properties. Liposomes were prepared by the thin-film hydration method followed by vesicle size reduction by sonication to the desired size, approximately 200 nm, and coated with chitosan (0.01, 0.03, 0.1, and 0.3%, w/v, respectively). The mucoadhesive properties of chitosan-coated liposomes were determined through their binding efficiency to mucin compared to non-coated liposomes. Non-coated liposomal suspensions were incorporated in chitosan hydrogels forming the liposomes-in-hydrogel formulations, which were further assessed for their texture properties in the presence of biological fluid simulants. The antibacterial effect of chitosan-coated liposomes (0.03%, 0.1% and 0.3%, w/v) and chitosan hydrogels (0.1% and 0.3%, w/w) on Staphylococcus epidermidis and Staphylococcus aureus was successfully confirmed.

BACKGROUND: The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. PRINCIPAL FINDINGS: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them α-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and α-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and α-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with α-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. CONCLUSIONS: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with α-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter.