Functional profiling of CHAP domain-containing peptidoglycan hydrolases of Staphylococcus aureus USA300 uncovers potential targets for anti-staphylococcal therapies.

The bacterial pathogen Staphylococcus aureus employs a thick cell wall for protection against physical and chemical insults. This wall requires continuous maintenance to ensure strength and barrier integrity, but also to permit bacterial growth and division. The main cell wall component is peptidoglycan. Accordingly, the bacteria produce so-called peptidoglycan hydrolases (PGHs) that cleave glycan strands to facilitate growth, cell wall remodelling, separation of divided cells and release of exported proteins into the extracellular milieu. A special class of PGHs contains so-called 'cysteine, histidine-dependent amidohydrolase/peptidase' (CHAP) domains. In the present study, we profiled the roles of 11 CHAP PGHs encoded by the core genome of S. aureus USA300 LAC. Mutant strains lacking individual CHAP PGHs were analysed for growth, cell morphology, autolysis, and invasion and replication inside human lung epithelial cells. The results show that several investigated CHAP PGHs contribute to different extents to extracellular and intracellular growth and replication of S. aureus, septation of dividing cells, daughter cell separation once the division process is completed, autolysis and biofilm formation. In particular, the CHAP PGHs Sle1 and SAUSA300_2253 control intracellular staphylococcal replication and the resistance to ß-lactam antibiotics like oxacillin. This makes the S. aureus PGHs in general, and the Sle1 and SAUSA300_2253 proteins in particular, attractive targets for future prophylactic or therapeutic anti-staphylococcal interventions. Alternatively, these cell surface-exposed enzymes, or particular domains of these enzymes, could be applied in innovative anti-staphylococcal therapies.

Time-resolved analysis of Staphylococcus aureus invading the endothelial barrier.

Staphylococcus aureus is a leading cause of infections world-wide. Once this pathogen has reached the bloodstream, it can invade different parts of the human body by crossing the endothelial barrier. Infected endothelial cells may be lysed by bacterial products, but the bacteria may also persist intracellularly, where they are difficult to eradicate with antibiotics and cause relapses of infection. Our present study was aimed at investigating the fate of methicillin resistant S. aureus (MRSA) isolates of the USA300 lineage with different epidemiological origin inside endothelial cells. To this end, we established two in vitro infection models based on primary human umbilical vein endothelial cells (HUVEC), which mimic conditions of the endothelium when infection occurs. For comparison, the laboratory strain S. aureus HG001 was used. As shown by flow cytometry and fluorescence- or electron microscopy, differentiation of HUVEC into a cell barrier with cell-cell junctions sets limits to the rates of bacterial internalization, the numbers of internalized bacteria, the percentage of infected cells, and long-term intracellular bacterial survival. Clear strain-specific differences were observed with the HG001 strain infecting the highest numbers of HUVEC and displaying the longest intracellular persistence, whereas the MRSA strains reproduced faster intracellularly. Nonetheless, all internalized bacteria remained confined in membrane-enclosed LAMP-1-positive lysosomal or vacuolar compartments. Once internalized, the bacteria had a higher propensity to persist within the differentiated endothelial cell barrier, probably because internalization of lower numbers of bacteria was less toxic. Altogether, our findings imply that intact endothelial barriers are more likely to sustain persistent intracellular infection.

Cytotoxicity of fractured quartz on THP-1 human macrophages: role of the membranolytic activity of quartz and phagolysosome destabilization.

The pathogenicity of quartz involves lysosomal alteration in alveolar macrophages. This event triggers the inflammatory cascade that may lead to quartz-induced silicosis and eventually lung cancer. Experiments with synthetic quartz crystals recently showed that quartz dust is cytotoxic only when the atomic order of the crystal surfaces is upset by fracturing. Cytotoxicity was not observed when quartz had as-grown, unfractured surfaces. These findings raised questions on the potential impact of quartz surfaces on the phagolysosomal membrane upon internalization of the particles by macrophages. To gain insights on the surface-induced cytotoxicity of quartz, as-grown and fractured quartz particles in respirable size differing only in surface properties related to fracturing were prepared and physico-chemically characterized. Synthetic quartz particles were compared to a well-known toxic commercial quartz dust. Membranolysis was assessed on red blood cells, and quartz uptake, cell viability and effects on lysosomes were assessed on human PMA-differentiated THP-1 macrophages, upon exposing cells to increasing concentrations of quartz particles (10-250 µg/ml). All quartz samples were internalized, but only fractured quartz elicited cytotoxicity and phagolysosomal alterations. These effects were blunted when uptake was suppressed by incubating macrophages with particles at 4 °C. Membranolysis, but not cytotoxicity, was quenched when fractured quartz was incubated with cells in protein-supplemented medium. We propose that, upon internalization, the phagolysosome environment rapidly removes serum proteins from the quartz surface, restoring quartz membranolytic activity in the phagolysosomes. Our findings indicate that the cytotoxic activity of fractured quartz is elicited by promoting phagolysosomal membrane alteration.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection.

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.