Thermosensitive PBP2a requires extracellular folding factors PrsA and HtrA1 for Staphylococcus aureus MRSA ß-lactam resistance.

Staphylococcus aureus is a major human pathogen and represents a clinical challenge because of widespread antibiotic resistance. Methicillin resistant Staphylococcus aureus (MRSA) is particularly problematic and originates by the horizontal acquisition of mecA encoding PBP2a, an extracellular membrane anchored transpeptidase, which confers resistance to ß-lactam antibiotics by allosteric gating of its active site channel. Herein, we show that dual disruption of PrsA, a lipoprotein chaperone displaying anti-aggregation activity, together with HtrA1, a membrane anchored chaperone/serine protease, resulted in severe and synergistic attenuation of PBP2a folding that restores sensitivity to ß-lactams such as oxacillin. Purified PBP2a has a pronounced unfolding transition initiating at physiological temperatures that leads to irreversible precipitation and complete loss of activity. The concordance of genetic and biochemical data highlights the necessity for extracellular protein folding factors governing MRSA ß-lactam resistance. Targeting the PBP2a folding pathway represents a particularly attractive adjuvant strategy to combat antibiotic resistance.

Vps13F links bacterial recognition and intracellular killing in Dictyostelium.

Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock-out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.

Missense mutations in PBP2A Affecting ceftaroline susceptibility detected in epidemic hospital-acquired methicillin-resistant Staphylococcus aureus clonotypes ST228 and ST247 in Western Switzerland archived since 1998.

The development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested the in vitro activity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 µg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 µg/ml; resistant [R], >1 µg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.

What can Dictyostelium bring to the study of Pseudomonas infections?

Bacterial infections are complex events. They are studied in a variety of simple model systems, using mammalian or non-mammalian hosts, all of which fail to reproduce fully the situation in infected patients. Each model presents a combination of conceptual, practical, and ethical advantages and disadvantages. In this review, we detail the use of Dictyostelium discoideum amoebae as a model to study Pseudomonas aeruginosa. More specifically, our aim is to explore what this additional model system can bring to our understanding of Pseudomonas infections. The study of interactions between Dictyostelium amoebae and Pseudomonas provides a view of the selection pressures exerted by environmental predators on Pseudomonas. It also represents a unique system to assess the virulence of very large numbers of Pseudomonas strains.

Role of magnesium and a phagosomal P-type ATPase in intracellular bacterial killing.

Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. The killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria.

Transmembrane domains control exclusion of membrane proteins from clathrin-coated pits.

Efficient sorting of proteins is essential to allow transport between intracellular compartments while maintaining their specific composition. During endocytosis, membrane proteins can be concentrated in endocytic vesicles by specific interactions between their cytoplasmic domains and cytosolic coat proteins. It is, however, unclear whether they can be excluded from transport vesicles and what the determinants for this sorting could be. Here, we show that in the absence of cytosolic sorting signals, transmembrane domains control the access of surface proteins to endosomal compartments. They act in particular by determining the degree of exclusion of membrane proteins from endocytic clathrin-coated vesicles. When cytosolic endocytosis signals are present, it is the combination of cytosolic and transmembrane determinants that ultimately controls the efficiency with which a given transmembrane protein is endocytosed.

Dictyostelium discoideum: a model host to measure bacterial virulence.

Dictyostelium amoebae have been used as a host model to measure virulence of a wide range of bacterial pathogens. The simple protocol described here takes advantage of the ability of Dictyostelium to grow and form plaques on a lawn of nonpathogenic bacteria but not on virulent bacteria. This assay can be modulated to measure the virulence of different bacterial pathogens. By adjusting various parameters such as cell numbers or media, a more quantitative measure of bacterial virulence can also be obtained. The entire procedure takes about 5 h to compete, and up to 2 further weeks may be required for plaques to form on the bacterial lawn.

A measure of endosomal pH by flow cytometry in Dictyostelium.

BACKGROUND: Dictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH. FINDINGS: We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells. CONCLUSION: Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH <3.5).