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.

Nosocomial transmission of tet(x3), bla NDM-1 and bla OXA-97-carrying Acinetobacter baumannii conferring resistance to eravacycline and omadacycline, the Netherlands, March to August 2021.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

Metabolic Profile of the Genome-Reduced Bacillus subtilis Strain IIG-Bs-27-39: An Attractive Chassis for Recombinant Protein Production.

The Gram-positive bacterium Bacillus subtilis is extensively used in the industry for the secretory production of proteins with commercial value. To further improve its performance, this microbe has been the subject of extensive genome engineering efforts, especially the removal of large genomic regions that are dispensable or even counterproductive. Here, we present the genome-reduced B. subtilis strain IIG-Bs-27-39, which was obtained through systematic deletion of mobile genetic elements, as well as genes for extracellular proteases, sporulation, flagella formation, and antibiotic production. Different from previously characterized genome-reduced B. subtilis strains, the IIG-Bs-27-39 strain was still able to grow on minimal media. We used this feature to benchmark strain IIG-Bs-27-39 against its parental strain 168 with respect to heterologous protein production and metabolic parameters during bioreactor cultivation. The IIG-Bs-27-39 strain presented superior secretion of difficult-to-produce staphylococcal antigens, as well as higher specific growth rates and biomass yields. At the metabolic level, changes in byproduct formation and internal amino acid pools were observed, whereas energetic parameters such as the ATP yield, ATP/ADP levels, and adenylate energy charge were comparable between the two strains. Intriguingly, we observed a significant increase in the total cellular NADPH level during all tested conditions and increases in the NAD+ and NADP(H) pools during protein production. This indicates that the IIG-Bs-27-39 strain has more energy available for anabolic processes and protein production, thereby providing a link between strain physiology and production performance. On this basis, we conclude that the genome-reduced strain IIG-Bs-27-39 represents an attractive chassis for future biotechnological applications.

From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins.

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis. IMPORTANCE: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade.

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

Post-translational secretion stress regulation in Bacillus subtilis is controlled by intra- and extracellular proteases.

The Gram-positive bacterium Bacillus subtilis is a prolific producer of industrial enzymes that are effectively harvested from the fermentation broth. However, the high capacity of B. subtilis for protein secretion has so far not been exploited to the full due to particular bottlenecks, including product degradation by extracellular proteases and counterproductive secretion stress responses. To unlock the Bacillus secretion pathway for difficult-to-produce proteins, various cellular interventions have been explored, including genome engineering. Our previous research has shown a superior performance of genome-reduced B. subtilis strains in the production of staphylococcal antigens compared to the parental strain 168. This was attributed, at least in part, to redirected secretion stress responses, including the presentation of elevated levels of the quality control proteases HtrA and HtrB that also catalyse protein folding. Here we show that this relates to the elimination of two homologous serine proteases, namely the cytosolic protease AprX and the extracellular protease AprE. This unprecedented posttranslational regulation of secretion stress effectors, like HtrA and HtrB, by the concerted action of cytosolic and extracellular proteases has important implications for the biotechnological application of microbial cell factories. In B. subtilis, this conclusion is underscored by extracellular degradation of the staphylococcal antigen IsaA by both AprX and AprE. Extracellular activity of the cytosolic protease AprX is remarkable since it shows that not only extracellular, but also intracellular proteases impact extracellular product levels. We therefore conclude that intracellular proteases represent new targets for improved recombinant protein production in microbial cell factories like B. subtilis.