Search

Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth, Staphylococcus aureus becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different S. aureus strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of S. aureus to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed., (© 2022. The Author(s).)

Comparative genomics has proven useful in exploring the biodiversity of phages and understanding phage-host interactions. This knowledge is particularly useful for phages infecting Streptococcus thermophilus, as they constitute a constant threat during dairy fermentations. Here, we explore the genetic diversity of S. thermophilus phages to identify genetic determinants with a signature for host specificity, which could be linked to the bacterial receptor genotype. A comparative genomic analysis was performed on 142 S. thermophilus phage genomes, 55 of which were sequenced in this study. Effectively, 94 phages were assigned to the group cos (DT1), 36 to the group pac (O1205), six to the group 5093, and six to the group 987. The core genome-based phylogeny of phages from the two dominating groups and their receptor binding protein (RBP) phylogeny corresponded to the phage host-range. A role of RBP in host recognition was confirmed by constructing a fluorescent derivative of the RBP of phage CHPC951, followed by studying the binding of the protein to the host strain. Furthermore, the RBP phylogeny of the cos group was found to correlate with the host genotype of the exocellular polysaccharide-encoding operon. These findings provide novel insights towards developing strategies to combat phage infections in dairies.

Bacterial cells are surrounded by cell wall, whose main component is peptidoglycan (PG), a macromolecule that withstands the internal turgor of the cell. PG composition can vary considerably between species. The Gram-positive pathogen Staphylococcus aureus possesses highly crosslinked PG due to the presence of cross bridges containing five glycines, which are synthesised by the FemXAB protein family. FemX adds the first glycine of the cross bridge, while FemA and FemB add the second and the third, and the fourth and the fifth glycines, respectively. Of these, FemX was reported to be essential. To investigate the essentiality of FemAB, we constructed a conditional S. aureus mutant of the femAB operon. Depletion of femAB was lethal, with cells appearing as pseudomulticellular forms that eventually lyse due to extensive membrane rupture. This deleterious effect was mitigated by drastically increasing the osmolarity of the medium, indicating that pentaglycine crosslinks are required for S. aureus cells to withstand internal turgor. Despite the absence of canonical membrane targeting domains, FemA has been shown to localise at the membrane. To study its mechanism of localisation, we constructed mutants in key residues present in the putative transferase pocket and the α6 helix of FemA, possibly involved in tRNA binding. Mutations in the α6 helix led to a sharp decrease in protein activity in vivo and in vitro but did not impair correct membrane localisation, indicating that FemA activity is not required for localisation. Our data indicates that, contrarily to what was previously thought, S. aureus cells do not survive in the absence of a pentaglycine cross bridge.

In living cells intracellular proteolysis is crucial for protein homeostasis, and ClpP proteases are conserved between eubacteria and the organelles of eukaryotic cells. In Staphylococcus aureus, ClpP associates to the substrate specificity factors, ClpX and ClpC forming two ClpP proteases, ClpXP and ClpCP. To address how individual ClpP proteases impact cell physiology, we constructed a S. aureus mutant expressing ClpX with an I 265 E substitution in the ClpP recognition tripeptide of ClpX. This mutant cannot degrade established ClpXP substrates confirming that the introduced amino acid substitution abolishes ClpXP activity. Phenotypic characterization of this mutant showed that ClpXP activity controls cell size and is required for growth at low temperature. Cells expressing the ClpX I265E variant, in contrast to cells lacking ClpP, are not sensitive to heat-stress and do not accumulate protein aggregates showing that ClpXP is dispensable for degradation of unfolded proteins in S. aureus. Consistent with this finding, transcriptomic profiling revealed strong induction of genes responding to protein folding stress in cells devoid of ClpP, but not in cells lacking only ClpXP. In the latter cells, highly upregulated loci include the urease operon, the pyrimidine biosynthesis operon, the betA-betB operon, and the pathogenicity island, SaPI5, while virulence genes were dramatically down-regulated.

β-lactam antibiotics target penicillin-binding proteins (PBPs) preventing peptidoglycan synthesis and this inhibition is circumvented in methicillin resistant Staphylococcus aureus (MRSA) strains through the expression of an additional PBP, named PBP2A. This enzyme is encoded by the mecA gene located within the Staphylococcal Chromosome Cassette mec (SCCmec) mobile genetic element, of which there are 12 types described to date. Previous investigations aimed at analysing the synergistic activity of two β-lactams, oxacillin and cefoxitin, found that SCCmec type IV community-acquired MRSA strains exhibited increased susceptibility to oxacillin in the presence of cefoxitin, while hospital-acquired MRSA strains were unaffected. However, it is not clear if these differences in β-lactam resistance are indeed a consequence of the presence of the different SCCmec types. To address this question, we have exchanged the SCCmec type I in COL (HA-MRSA) for the SCCmec type IV from MW2 (CA-MRSA). This exchange did not decrease the resistance of COL against oxacillin and cefoxitin, as observed in MW2, indicating that genetic features residing outside of the SCCmec element are likely to be responsible for the discrepancy in oxacillin and cefoxitin synergy against these MRSA strains.