A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE‐like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)‐PCR analysis method established. LA‐PCR products of 13–17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb‐harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq‐harboring SaPIj11 and non‐superantigen‐harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA‐PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.     

Virus Satellites Drive Viral Evolution and Ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts.     

Control of Staphylococcus aureus pathogenicity island excision

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15–17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small‐headed phage‐like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI‐encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI‐coded Xis is absolutely required for recombination between the SaPI att_L and att_R sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.     

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage–SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.     

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo,Japan

Staphylococcal food poisoning (SFP), one of the commonest food‐borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin‐encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.     

Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in staphylococci

Although mobile genetic elements have a crucial role in spreading pathogenicity-determining genes among bacterial populations, environmental and genetic factors involved in the horizontal transfer of these genes are largely unknown. Here we show that SaPIbov1, a Staphylococcus aureus pathogenicity island that belongs to the growing family of these elements that are found in many strains, is induced to excise and replicate after SOS induction of at least three different temperate phages, 80alpha, phi11 and phi147, and is then packaged into phage-like particles and transferred at high frequency. SOS induction by commonly used fluoroquinolone antibiotics, such as ciprofloxacin, also results in replication and high-frequency transfer of this element, as well as of SaPI1, the prototypical island of S. aureus, suggesting that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors. Although the strains containing these prophages do not normally contain SaPIs, we have found that RF122-1, the original SaPIbov1-containing clinical isolate, contains a putative second pathogenicity island that is replicated after SOS induction, by antibiotic treatment, of the prophage(s) present in the strain. Although SaPIbov1 is not induced to replicate after SOS induction in this strain, it is transferred by the antibiotic-activated phages. We conclude that SOS induction by therapeutic agents can promote the spread of staphylococcal virulence genes.     

Unravelling bacteriophage ϕ11 requirements for packaging and transfer of mobile genetic elements in Staphylococcus aureus

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)‐encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ?11, used as a model for the Siphoviridae phages infecting Gram‐positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage‐encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ?11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage‐mediated MGEs transfer.     

Fatal S. aureus hemorrhagic pneumonia: genetic analysis of a unique clinical isolate producing both PVL and TSST-1

In 2008, an unusual strain of methicillin-sensitive Staphylococcus aureus (MSSA68111), producing both Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1), was isolated from a fatal case of necrotizing pneumonia. Because PVL/TSST-1 co-production in S. aureus is rare, we characterized the molecular organization of these toxin genes in strain 68111. MSSA68111 carries the PVL genes within a novel temperate prophage we call ФPVLv68111 that is most similar, though not identical, to phage ФPVL--a phage type that is relatively rare worldwide. The TSST-1 gene (tst) in MSSA68111 is carried on a unique staphylococcal pathogenicity island (SaPI) we call SaPI68111. Features of SaPI68111 suggest it likely arose through multiple major recombination events with other known SaPIs. Both ФPVLv68111 and SaPI68111 are fully mobilizable and therefore transmissible to other strains. Taken together, these findings suggest that hypervirulent S. aureus have the potential to emerge worldwide.     

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.     

Methicillin Susceptible Staphylococcus aureus (MSSA) of Clonal Complex CC398, t571 from Infections in Humans Are Still Rare in Germany

Methicillin-susceptible Staphylococcus aureus (MSSA) attributed to clonal complex (CC) 398 and exhibiting spa-type t571 received attention in Europe and in the USA for being associated with severe infections in humans. As this spa-type is exhibited by livestock-associated (LA) Methicillin-resistant S. aureus (MRSA) as well, it is important to discriminate LA- and human-derived strains by easy to perform, PCR-based methods. MSSA t571 contain phage int3 carrying scn and chp, whereas LA-MRSA t571 lack these markers. In contrast, pathogenicity island SaPIbov5 (detected by PCR bridging vwbbov and scn) is contained by LA-MRSA t571 and absent in the human MSSA subpopulation. Furthermore, MSSA t571 contain erm(T), the particular genomic arrangement of which was assessed by a PCR bridging erm(T) and the adjacent transposase gene. MSSA t571 are rare so far in Germany among isolates from infections in humans (0.14%) as well as among isolates from nasal colonization (0.13%). LA-MRSA t571 are also infrequent among MRSA isolated from carriage at admission to hospitals (0.1%) and also among isolates from infections in humans (0.013%).     

Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome

Staphylococcus aureus causes disease in humans and a wide array of animals. Of note, S. aureus mastitis of ruminants, including cows, sheep, and goats, results in major economic losses worldwide. Extensive variation in genome content exists among S. aureus pathogenic clones. However, the genomic variation among S. aureus strains infecting different animal species has not been well examined. To investigate variation in the genome content of human and ruminant S. aureus, we carried out whole-genome PCR scanning (WGPS), comparative genomic hybridizations (CGH), and the directed DNA sequence analysis of strains of human, bovine, ovine, and caprine origin. Extensive variation in genome content was discovered, including host- and ruminant-specific genetic loci. Ovine and caprine strains were genetically allied, whereas bovine strains were heterogeneous in gene content. As expected, mobile genetic elements such as pathogenicity islands and bacteriophages contributed to the variation in genome content between strains. However, differences specific for ruminant strains were restricted to regions of the conserved core genome, which contained allelic variation in genes encoding proteins of known and unknown function. Many of these proteins are predicted to be exported and could play a role in host-pathogen interactions. The genomic regions of difference identified by the whole-genome approaches adopted in the current study represent excellent targets for studies of the molecular basis of S. aureus host adaptation.     

Involvement of caspase‐1 in inflammasomes activation and bacterial clearance in S. aureus‐infected osteoblast‐like MG‐63 cells

Staphylococcus aureus, a versatile Gram‐positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase‐1 that proteolytically matures and promotes the secretion of mature IL‐1β and IL‐18. The role of inflammasomes and caspase‐1 in the secretion of mature IL‐1β and in the defence of S. aureus‐infected osteoblasts has not yet been fully investigated. We show here that S. aureus‐infected osteoblast‐like MG‐63 but not caspase‐1 knock‐out CASP1 ^?/?MG‐63 cells, which were generated using CRISPR‐Cas9 technology, activate the inflammasome as monitored by the release of mature IL‐1β. The effect was strain‐dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes‐related IL‐1β production. Furthermore, we found that the lack of caspase‐1 in CASP1 ^?/?MG‐63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 ^?/? MG‐63 compared to wild‐type cells. Our results demonstrate that osteoblast‐like MG‐63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase‐1 in bacterial clearance.     

Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1–DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD′s presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep′s activities as initiators and as helicases.     

Use of loop‐mediated isothermal amplification to detect six groups of pathogens causing secondary lower respiratory bacterial infections in horses

Microbial substitution occasionally occurs following the administration of antimicrobials to horses that have pneumonia or pleuropneumonia. Four specific loop‐mediated isothermal amplification (LAMP) assays were developed to detect some equine respiratory pathogens, namely strains of the Bacteroides–Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Staphylococcus aureus. These four LAMP assays and two previously published LAMP assays targeting Escherichia coli or Pseudomonas aeruginosa were used on clinical respiratory specimens and a high accordance found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials.     

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T = 7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.     

Multiple Ligands of von Willebrand Factor-binding Protein (vWbp) Promote Staphylococcus aureus Clot Formation in Human Plasma

Staphylococcus aureus secretes coagulase (Coa) and von Willebrand factor-binding protein (vWbp) to activate host prothrombin and form fibrin cables, thereby promoting the establishment of infectious lesions. The D1-D2 domains of Coa and vWbp associate with, and non-proteolytically activate prothrombin. Moreover, Coa encompasses C-terminal tandem repeats for binding to fibrinogen, whereas vWbp has been reported to associate with von Willebrand factor and fibrinogen. Here we used affinity chromatography with non-catalytic Coa and vWbp to identify the ligands for these virulence factors in human plasma. vWbp bound to prothrombin, fibrinogen, fibronectin, and factor XIII, whereas Coa co-purified with prothrombin and fibrinogen. vWbp association with fibrinogen and factor XIII, but not fibronectin, required prothrombin and triggered the non-proteolytic activation of FXIII in vitro. Staphylococcus aureus coagulation of human plasma was associated with the recruitment of prothrombin, FXIII, and fibronectin as well as the formation of cross-linked fibrin. FXIII activity in staphylococcal clots could be attributed to thrombin-dependent proteolytic activation as well as vWbp-mediated non-proteolytic activation of FXIII zymogen.