The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

Toxin–antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin–antitoxin system (pemIK_Sa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemK_Sa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemI_Sa) inhibits toxin activity by physical interaction. Although the toxin–antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIK_Sa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin–antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin–antitoxin systems.

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.

Staphylococcus aureus is a widespread, opportunistic pathogen that causes community and hospital acquired infections. Its high pathogenicity is driven by multifactorial and complex mechanisms determined by the ability of the bacterium to express a wide variety of virulence factors. The proteome secreted into extracellular milieu is a rich reservoir of such factors which include mainly nonenzymatic toxins and enzymes. Simultaneously, membrane proteins, membrane-cell wall interface proteins and cell wall-associated proteins also strongly influence staphylococcal virulence. Proteomics shows a great potential in exploring the role of the extracellular proteome in cell physiology, including the pathogenic potential of particular strains of staphylococci. In turn, understanding the bacterial physiology including the interconnections of particular factors within the extracellular proteomes is a key to the development of the ever needed, novel antibacterial strategies. Here, we briefly overview the latest applications of gel-based and gel-free proteomic techniques in the identification of the virulence factors within S. aureus secretome and surfacome. Such studies are of utmost importance in understanding the host-pathogen interactions, analysis of the role of staphylococcal regulatory systems and also the detection of posttranslational modifications emerging as important modifiers of the infection process.