The Spx stress regulator confers high-level ß-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA has acquired resistance to next-generation ß-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators, which, through poorly described mechanisms, enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased ß-lactam MICs up to 16-fold and transformed MRSA cells with low levels of resistance to being homogenously highly resistant to ß-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPR interference (CRISPRi) to knock down spx transcription, we unambiguously linked hyper-resistance to the accumulation of Spx. Spx was previously proposed to be essential; however, our data suggest that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in ß-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane, including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox-sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum ß-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

Possible interactions between dietary fibres and 5-aminosalicylic acid [corrected].

BACKGROUND: Potentially, a binding of 5-aminosalicylic acid (5-ASA) to dietary fibres could reduce the systemic absorption and increase the intraluminal amount [corrected]. The purposes of the study were to investigate if: (1) dietary fibres can bind 5-ASA in vitro, and (2) consumption of dietary fibres is related to disease activity in patients with ulcerative colitis (UC) treated with 5-ASA. METHODS: In vitro: 15 g of Ispaghula Husk, wheat bran, citrus-pectin, or wheat flour were incubated in a 37°C buffered solutions of 5-ASA (1 g/l) for 3 hours at pH 6 and 7. The concentrations of 5-ASA were determined before and after the incubation using HPLC. In vivo: patients with UC were interviewed two to three times during 6 months. The fibre consumption was estimated and related to the disease activity (CAI, CRP, Faecal-calprotectin) and quality of life (IBDQ). RESULTS: In vitro: 5-ASA was bound to Ispaghula Husk (5.3-10.0 mg/g) and wheat bran (4.6-5.5 mg/g), and to a minor degree to citrus-pectin. No differences were found in relation to pH. In vivo: 29 patients completed the scheduled interviews. No significant changes in fibre consumption were observed over time; however, patients consuming a diet high in fibre (>20 g/day) had significantly lower CRP (p <0.01) and faecal-calprotectin (p <0.01) than those consuming less fibre (<20 g/dg). CONCLUSIONS: Patients with a high intake of fibre had a lower disease activity than those with low intake. Ispaghula Husk bound 5-ASA in vitro, independent of pH. The effect might be clinically relevant in patients with UC treated with 5-ASA.