A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non‐professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre‐incubation of endothelial cells with recombinant Atl dose‐dependently inhibited internalization. As putative Atl‐host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti‐Hsc70 antibodies. In conclusion, this novel Atl‐ or AtlE‐mediated internalization mechanism may represent a ‘back‐up’ mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase‐negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

EGFR/FAK and c‐Src signalling pathways mediate the internalisation of Staphylococcus aureus by osteoblasts

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor‐focal adhesion kinase (FAK), phosphor‐epidermal growth factor receptor (EGFR) and phosphor‐c‐Src in a time‐dependent way, and blocking EGFR/FAK or c‐Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c‐Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.     

Functional basis for complement evasion by staphylococcal superantigen‐like 7

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen‐like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA‐FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b‐9‐mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal‐induced C5a‐mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA‐binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a‐induced phagocytosis of S. aureus and oxidative burst in an in vitro whole‐blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a‐driven influx of neutrophils in mouse peritoneum.     

Exploiting dominant‐negative toxins to combat Staphylococcus aureus pathogenesis

Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta‐barrel, bi‐component, pore‐forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine‐rich motifs within the membrane‐penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine‐rich motifs exhibit dominant‐negative inhibitory effects toward their wild‐type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant‐negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant‐negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild‐type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi‐component dominant‐negative toxins and their potential as novel therapeutics to combat S. aureus infection.     

A role for host cell exocytosis in InlB‐mediated internalisation of Listeria monocytogenes

The bacterial surface protein InlB mediates internalisation of Listeria monocytogenes into human cells through interaction with the host receptor tyrosine kinase, Met. InlB‐mediated entry requires localised polymerisation of the host actin cytoskeleton. Apart from actin polymerisation, roles for other host processes in Listeria entry are unknown. Here, we demonstrate that exocytosis in the human cell promotes InlB‐dependent internalisation. Using a probe consisting of VAMP3 with an exofacial green fluorescent protein tag, focal exocytosis was detected during InlB‐mediated entry. Exocytosis was dependent on Met tyrosine kinase activity and the GTPase RalA. Depletion of SNARE proteins by small interfering RNA demonstrated an important role for exocytosis in Listeria internalisation. Depletion of SNARE proteins failed to affect actin filaments during internalisation, suggesting that actin polymerisation and exocytosis are separable host responses. SNARE proteins were required for delivery of the human GTPase Dynamin 2, which promotes InlB‐mediated entry. Our results identify exocytosis as a novel host process exploited by Listeria for infection.     

Staphylococcus aureus protects its immune‐evasion proteins against degradation by neutrophil serine proteases

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti‐staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune‐evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune‐escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

What determines nasal carriage of Staphylococcus aureus?

Nasal carriage of Staphylococcus aureus is an important risk factor for infection by this organism in both community and hospital settings; this article reviews the role of host and bacterial factors in carriage. A host genetic influence appears likely but the phenotypic determinants are unknown. Possibilities include variability in host adhesins, immune response or secretion of antimicrobial molecules. Colonization resistance by S. aureus, together with the observation that persistent carriers often carry a single strain whereas intermittent carriers can be colonized with unrelated strains over time, suggests that bacterial factors could also be involved.     

Helicobacter pylori adhesins: review and perspectives

It is highly unlikely that chronic infection with H. pylori could occur in the absence of adhesin–host cell interactions. Also, there is no evidence that any of the serious outcomes of H. pylori infection such as gastric and duodenal ulcers, gastric cancer or mucosa‐associated lymphoid tissue (MALT) lymphoma could occur without prior colonization of the gastric epithelium mediated by H. pylori adhesins. H. pylori is highly adaptable, as evidenced by the fact that it can occupy a single host for decades. An important facet of this adaptability is its ability to physically interact with various types of host cells and also with host mucins and extracellular matrix proteins using a number of different adhesins displaying a variety of unique receptor specificities. Thus it is highly unlikely that any one particular H. pylori adhesin will ever be proven responsible for a particular outcome such as duodenal ulcer, MALT lymphoma, or adenocarcinoma. Also, while the search for additional H. pylori adhesins should and certainly will continue, we suggest that the scope of this effort should be expanded to include investigations into the patterns of expression and interaction between individual outer membrane proteins. Which of the numerous H. pylori outer membrane proteins (OMPs) actually function as adhesins (i.e., have receptor‐binding sites) and which OMPs are simply necessary for optimal display of the adhesive OMPs? There are many other important questions about H. pylori adhesins waiting to be answered. For example, which adhesins are responsible for loose adherence to host cells and which adhesins are responsible for intimate, or membrane‐to‐membrane, adherence, and do these adhesins normally work in concert or in a sequential fashion? Also, is a specific type of adhesin necessary for type IV protein translocation into host cells and, if so, is adhesin expression coregulated with the effector protein export?     

Escherichia coli K‐12 possesses multiple cryptic but functional chaperone–usher fimbriae with distinct surface specificities

Commensal and pathogenic Escherichia coli adherence to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium, 34% of the genes in the E. coli K‐12 genome have no known function. We hypothesized that some of them may correspond to functional adhesins. We characterized E. coli K‐12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organizational homologies to type 1 fimbriae exported by the chaperone/usher pathway. We showed that, although these operons are poorly expressed under laboratory conditions, six of them are nevertheless functional when expressed, and promote adhesion to abiotic and/or epithelial cell surfaces. While the studied fimbriae display different binding specificities, we obtained evidence of synergy/interference with other adhesins such as Ag43 or type 1 fimbriae. We showed that their expression is under the negative control of H‐NS and, except for yad, subjected to cAMP receptor protein‐mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbriae adhesins whose expression following environmental modifications could contribute to E. coli's ability to adhere to and colonize a wide diversity of surfaces in its various ecological niches.     

Stimulation of delta opioid receptors located in substantia nigra reticulata but not globus pallidus or striatum restores motor activity in 6‐hydroxydopamine lesioned rats: new insights into the role of delta receptors in parkinsonism

The delta opioid peptide (DOP) receptor has been proposed as a target in the symptomatic therapy of Parkinson’s disease. However, the circuitry underlying the antiparkinsonian action of DOP receptor agonists and their site of action have never been adequately investigated. Systemic administration of the DOP receptor agonist (+)‐4‐[(αR)‐α‐(2S,5R)‐allyl‐2,5‐dimethyl‐1‐piperazinyl)‐3‐methoxy‐benzyl]‐N‐N‐diethylbenzamide (SNC‐80) attenuated akinesia/bradykinesia and improved motor activity in 6‐hydroxydopamine hemilesioned rats. Opposite effects were produced by the selective DOP receptor antagonist naltrindole (NTD), suggesting that endogenous enkephalins tonically sustain movement under parkinsonian conditions. Microdialysis revealed that SNC‐80 reduced GABA release in globus pallidus (GP) while NTD elevated it. Moreover, SNC‐80 reduced GABA and glutamate release in substantia nigra reticulata (SNr) whereas NTD reduced GABA without affecting glutamate release. The bar test coupled to microdialysis showed that perfusion with NTD in SNr but not GP or striatum prevented the antiakinetic effect of systemic SNC‐80 and its neurochemical correlates. Consistently, microinjections of SNC‐80 into SNr or bicuculline in GP attenuated parkinsonian‐like symptoms while SNC‐80 microinjections in GP or striatum were ineffective. This study demonstrates that nigral DOP receptors mediate antiparkinsonian actions of SNC‐80 and challenges the common view that DOP receptor agonists solely attenuate parkinsonism via pallidal mechanisms.     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10‐encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10‐encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA‐binding fold, and prgB‐prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA‐binding regulators that act to mitigate toxicity accompanying overproduction of PrgB‐like adhesins in E. faecalis and other clinically‐important Gram‐positive species.     

Staphylococcal phosphatidylinositol‐specific phospholipase C potentiates lung injury via complement sensitisation

Staphylococcus aureus is frequently isolated from patients with community‐acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol‐specific phospholipase C (PI‐PLC); however, the role of PI‐PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI‐PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol‐anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI‐PLC‐positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc‐isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild‐type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc^? mutant. Following treatment with cobra venom factor to deplete complement, the wild‐type strain with PI‐PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI‐PLC‐positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI‐PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.     

Evaluation of lytic activity of staphylococcal bacteriophage Sb‐1 against freshly isolated clinical pathogens

In recent decades the increase in antibiotic‐resistant bacterial strains has become a serious threat to the treatment of infectious diseases. Drug resistance of Staphylococcus aureus has become a major problem in hospitals of many countries, including developed ones. Today the interest in alternative remedies to antibiotics, including bacteriophage treatment, is gaining new ground. Here, we describe the staphylococcal bacteriophage Sb‐1 – a key component of therapeutic phage preparation that was successfully used against staphylococcal infections during many years in the Former Soviet Union. This phage still reveals a high spectrum of lytic activity in vitro against freshly isolated, genetically different clinical samples (including methicillin‐resistant S. aureus) obtained from the local hospitals, as well as the clinics from different geographical areas. The sequence analyses of phage genome showed absence of bacterial virulence genes. A case report describes a promising clinical response after phage application in patient with cystic fibrosis and indicates the efficacy of usage of Sb‐1 phage against various staphylococcal infections.     

Transfer of the methicillin resistance genomic island among staphylococci by conjugation

Methicillin resistance creates a major obstacle for treatment of Staphylococcus aureus infections. The resistance gene, mecA, is carried on a large (20 kb to > 60 kb) genomic island, staphylococcal cassette chromosome mec (SCCmec), that excises from and inserts site‐specifically into the staphylococcal chromosome. However, although SCCmec has been designated a mobile genetic element, a mechanism for its transfer has not been defined. Here we demonstrate the capture and conjugative transfer of excised SCCmec. SCCmec was captured on pGO400, a mupirocin‐resistant derivative of the pGO1/pSK41 staphylococcal conjugative plasmid lineage, and pGO400::SCCmec (pRM27) was transferred by filter‐mating into both homologous and heterologous S. aureus recipients representing a range of clonal complexes as well as S. epidermidis. The DNA sequence of pRM27 showed that SCCmec had been transferred in its entirety and that its capture had occurred by recombination between IS257/431 elements present on all SCCmec types and pGO1/pSK41 conjugative plasmids. The captured SCCmec excised from the plasmid and inserted site‐specifically into the chromosomal att site of both an isogenic S. aureus and a S. epidermidis recipient. These studies describe a means by which methicillin resistance can be environmentally disseminated and a novel mechanism, IS‐mediated recombination, for the capture and conjugative transfer of genomic islands.     

Nfu facilitates the maturation of iron‐sulfur proteins and participates in virulence in Staphylococcus aureus

The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron‐sulfur (Fe‐S) clusters, which are required for functional Fe‐S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe‐S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe‐S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe‐S cluster carrier, which aids in the maturation of Fe‐S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non‐incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe‐S cluster metabolism as an attractive antimicrobial target.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .