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.

SummaryBacterial 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.

Staphylococcus aureus is a major pathogen worldwide and can provoke a range of diseases that range from relatively minor to life-threatening. It is the most common etiological agent of surgical site infections and ventilator-associated pneumonia. Of particular concern are infections arising from encounters with antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA). Methicillin resistance in S. aureus is dependent upon the acquisition of the mecA gene, which encodes penicillin-binding protein 2A (PBP2A), which is refractory to inhibition by methicillin and numerous other ␤-lactam antibiotics (1).MRSA is widely recognized as a serious threat to public health, and new antibiotics are urgently needed (2). Penicillin-binding proteins (PBPs), which are bacterial enzymes catalyzing the last steps of cell wall biosynthesis, have long been used as lethal targets for ␤-lactam antibiotics. Ceftaroline, a novel ␤-lactam broadspectrum cephalosporin, also binds to PBPs (3); however, an exceptional characteristic of ceftaroline is that it also binds to and inhibits PBP2A, effectively blocking the principal ␤-lactam resistance determinant of MRSA strains. Accordingly, numerous studies have attested that ceftaroline shows robust in vitro activity against MRSA strains (4-8).Neither clinical nor in vitro studies have detected high percentages or rapid development of ceftaroline resistance (9). Nevertheless, some rare resistant strains have been reported (10, 11) (EUCAST MICs, Ͼ1 g/ml or CLSI MIC, Ն4 g/ml). To our knowledge, only a few studies have been conducted that identified PBP2A mutations as being correlated with a reduction in sensitivity to ceftaroline (11,12). Some of these PBP2A mutations (N146K and E150K) correlated with decreased affinity to ceftaroline (13) and were found to be located in the recently discovered allosteric site that is considerably distant from the transpeptidase active site domain (13,14). Interestingly, as shown by Otero and coworkers (14) using X-ray crystallographic analysis, ceftaroline is capable of binding both the PBP2A allosteric and the DD-transpeptidase domains. In the proposed model, noncovalent ceftaroline binding to the allosteric site might influence accessibility to the PBP2A active site by a second ceftaroline molecule over a considerable spatial distance by an intricate network of salt bridges and conformational changes. Consequently, mutations in the PBP2A alloste-

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.

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.