Ladan Khodaparast

Vaccination with SesC Decreases Staphylococcus epidermidis Biofilm Formation

ABSTRACT The increased use of medical implants has resulted in a concomitant rise in device-related infections. The majority of these infections are caused by Staphylococcus epidermidis biofilms. Immunoprophylaxis and immunotherapy targeting in vivo-expressed, biofilm-associated, bacterial cell surface-exposed proteins are promising new approaches to prevent and treat biofilm-related infections, respectively. Using an in silico procedure, we identified 64 proteins that are predicted to be S. epidermidis surface exposed (Ses), of which 36 were annotated as (conserved) hypothetical. Of these 36 proteins, 5 proteins—3 LPXTG motif-containing proteins (SesL, SesB, and SesC) and 2 of the largest ABC transporters (SesK and SesM)—were selected for evaluation as vaccine candidates. This choice was based on protein size, number of antigenic determinants, or the established role in S. epidermidis biofilm formation of the protein family to which the candidate protein belongs. Anti-SesC antibodies exhibited the greatest inhibitory effect on S. epidermidis biofilm formation in vitro and on colonization and infection in a mouse jugular vein catheter infection model that includes biofilms and organ infections. Active vaccination with a recombinant truncated SesC inhibited S. epidermidis biofilm formation in a rat model of subcutaneous foreign body infection. Antibodies to SesC were shown to be opsonic by an in vitro opsonophagocytosis assay. We conclude that SesC is a promising target for antibody mediated strategies against S. epidermidis biofilm formation.

De novo design of a biologically active amyloid

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science, this issue p. 10.1126/science.aah4949 A designed peptide drives a protein that does not usually aggregate to form amyloids. INTRODUCTION It has been shown that most proteins possess amyloidogenic segments. However, only about 30 human proteins are known to be involved in amyloid-associated pathologies, and it is still not clear what determines amyloid toxicity in these diseases. We investigated whether an endogenously expressed protein that contains sequences with known amyloidogenic segments, but is not known to aggregate either under normal or pathological conditions, can be induced to do so by seeding it with a peptide comprising the protein’s own amyloidogenic fragment. We chose to target the protein vascular endothelial growth factor receptor 2 (VEGFR2) because it has well-characterized biological function and so could provide a model system with which to investigate the relationship between protein loss of function and amyloid toxicity in different cellular contexts. RATIONALE The capacity of the amyloid conformation of disease proteins to catalyze their own amyloid conversion demonstrates the sequence specificity of amyloid assembly. Because the core of amyloids consists of short amyloidogenic sequence fragments, we hypothesized that a short amyloidogenic protein sequence of VEGFR2, a protein normally not associated with protein aggregation, should be able to interact with and specifically induce the aggregation of VEGFR2, resulting in its functional knockdown. We used TANGO, an algorithm that predicts aggregation-prone sequences, to identify potential amyloidogenic fragments in VEGFR2. We synthesized these fragments as a tandem repeat in a peptide framework in which each unit is flanked by charged residues and coupled by a short peptide linker. The thinking behind this design was that the tandem repeats would promote the formation of diffusable soluble oligomeric aggregates, whereas the charged residues would kinetically stabilize these oligomers and reduce the rate of insoluble fibril formation. RESULTS By screening for loss of function, we identified one peptide, termed “vascin,” that was highly potent at inhibiting VEGFR2. This sequence was derived from the translocation signaling sequence of VEGFR2. We found that vascin is an amyloidogenic peptide that readily forms small β-structured oligomers, ranging from dimers to nonamers, that slowly convert to amyloid fibrils. When added to cell culture medium, these oligomers are efficiently absorbed by the cell, where they interact with and promote the aggregation and partial degradation of nascent VEGFR2. Vascin aggregation does not induce the aggregation of known disease amyloids. Neither do vascin oligomers affect the function of the related EGF receptor or the surface translocation of other receptors. We found vascin only to be toxic to cells that are dependent on VEGFR2 function, suggesting that toxicity is due to loss of VEGFR2 function and not to vascin aggregation or vascin-induced VEGFR2 aggregation. Consistent with this, we found that vascin is active in vivo and could reduce tumor growth in a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor model in mice but is not intrinsically toxic to other tissues. CONCLUSION We found that a short amyloidogenic protein fragment can induce the aggregation of a protein normally not associated with amyloidosis in a manner that recapitulates key biophysical and biochemical characteristics of natural amyloids. In addition, we found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, rather than being generic, amyloid toxicity here appears to be both protein-specific and conditional on a requirement for VEGFR2 protein function. A synthetic amyloid peptide induces aggregation. We designed vascin, a synthetic amyloid peptide based on an amyloidogenic fragment of the signal peptide of VEGFR2. Vascin forms prefibrillar oligomers that penetrate mammalian cells and interacts with the nascent VEGFR2 protein, resulting in its aggregation and functional knockdown. [Composition includes parts of an image from iStock.com/luismmolina.] Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional—determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.Aggregation is sequence-specific and nucleated by short aggregating protein segments (APR). Here authors use a multidisciplinary approach to show that in E.coli some frequently occurring APRs lead to protein aggregation and ultimately bacterial cell death, which could serve as antibacterial strategy.

Ses proteins as possible targets for vaccine development against Staphylococcus epidermidis infections

OBJECTIVES The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. METHODS Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytotic capacity of these IgGs. RESULTS Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonization with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. CONCLUSIONS Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains.

sesC as a genetic marker for easy identification of Staphylococcus epidermidis from other isolates.

Staphylococcus epidermidis is one of the major concerns with respect to hospital-acquired infections. Therefore, a rapid and easy method to identify at species level S. epidermidis isolates out of a broad range of bacteria is necessary. Based on earlier studies, the sesC gene encoding a S. epidermidis surface protein revealed to be a highly conserved gene in this species. By means of an easy and inexpensive PCR assay, the presence of sesC was checked in 438 clinical staphylococcal isolates. Results showed that sesC is specifically present in all S. epidermidis. In conclusion, the sesC gene can be exploited as a genetic marker in order to distinguish S. epidermidis from other isolates.