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Staphylococcus aureus possesses a large arsenal of immune-modulating factors, enabling it to bypass the immune system’s response. Here, we demonstrate that the acid phosphatase SapS is secreted during macrophage infection and promotes its intracellular survival in this type of immune cell. In animal models, the SA564 sapS mutant demonstrated a significantly lower bacterial burden in liver and renal tissues of mice at four days post infection in comparison to the wild type, along with lower pathogenicity in a zebrafish infection model. The SA564 sapS mutant elicits a lower inflammatory response in mice than the wild-type strain, while S. aureus cells harbouring a functional sapS induce a chemokine response that favours the recruitment of neutrophils to the infection site. Our in vitro and quantitative transcript analysis show that SapS has an effect on S. aureus capacity to adapt to oxidative stress during growth. SapS is also involved in S. aureus biofilm formation. Thus, this study shows for the first time that SapS plays a significant role during infection, most likely through inhibiting a variety of the host’s defence mechanisms.

The rise of antimicrobial resistance poses a severe threat to public health. The natural product chlorotonil was identified as a new antibiotic targeting multidrug resistant Gram-positive pathogens and Plasmodium falciparum. Although chlorotonil shows promising activities, the scaffold is highly lipophilic and displays potential biological instabilities. Therefore, we strived towards improving its pharmaceutical properties by semisynthesis. We demonstrated stereoselective epoxidation of chlorotonils and epoxide ring opening in moderate to good yields providing derivatives with significantly enhanced solubility. Furthermore, in vivo stability of the derivatives was improved while retaining their nanomolar activity against critical human pathogens (e.g. methicillin-resistant Staphylococcus aureus and P. falciparum). Intriguingly, we showed further superb activity for the frontrunner molecule in a mouse model of S. aureus infection.

Staphylococcus aureus continues to be a public health threat, especially in hospital settings. Studies aimed at deciphering the molecular and cellular mechanisms that underlie pathogenesis, host adaptation, and virulence are required to develop effective treatment strategies. Numerous host-pathogen interactions were found to be dependent on phosphatases-mediated regulation. This study focused on the analysis of the role of the low-molecular weight phosphatase PtpB, in particular, during infection. Deletion of ptpB in S. aureus strain SA564 significantly reduced the capacity of the mutant to withstand intracellular killing by THP-1 macrophages. When injected into normoglycemic C57BL/6 mice, the SA564 ΔptpB mutant displayed markedly reduced bacterial loads in liver and kidney tissues in a murine S. aureus abscess model when compared to the wild type. We also observed that PtpB phosphatase-activity was sensitive to oxidative stress. Our quantitative transcript analyses revealed that PtpB affects the transcription of various genes involved in oxidative stress adaptation and infectivity. Thus, this study disclosed first insights into the physiological role of PtpB during host interaction allowing us to link phosphatase-dependent regulation to oxidative bacterial stress adaptation during infection.

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram-positive bacteria, catabolite control protein A (CcpA) and the histidine-containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation.

Chronic wounds infected with pathogens such as Staphylococcus aureus represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP). In this work, we describe the establishment of an in vivo wound infection model based on the pathogen S. aureus and hairless Crl:SKH1-Hrhr (SKH1) mice. The model proved to be a valuable preclinical tool to study selected RNAP inhibitors after topical application. While rifampicin showed a reduction in the loss of body weight induced by the bacteria, an acceleration of wound healing kinetics, and a reduced number of colony forming units in the wound, the ureidothiophene-2-carboxylic acid 1 was inactive under in vivo conditions, probably due to strong plasma protein binding. The cocrystal structure of compound 1 with RNAP, that we hereby also present, will be of great value for applying appropriate structural modifications to further optimize the compound, especially in terms of plasma protein binding.

Drug delivery systems are promising for targeting antibiotics directly to infected tissues. To reach intracellular Staphylococcus aureus and Mycobacterium abscessus, we encapsulated clarithromycin in PLGA nanocapsules, suitable for aerosol delivery by nebulization of an aqueous dispersion. Compared to the same dose of free clarithromycin, nanoencapsulation reduced 1000 times the number of intracellular S. aureus in vitro. In RAW cells, while untreated S. aureus was located in acidic compartments, the treated ones were mostly situated in non-acidic compartments. Clarithromycin-nanocapsules were also effective against M. abscessus (70-80% killing efficacy). The activity of clarithromycin-nanocapsules against S. aureus was also confirmed in vivo, using a murine wound model as well as in zebrafish. The permeability of clarithromycin-nanocapsules across Calu-3 monolayers increased in comparison to the free drug, suggesting an improved delivery to sub-epithelial tissues. Thus, clarithromycin-nanocapsules are a promising strategy to target intracellular S. aureus and M. abscessus.

Secretion of bacterial signaling proteins and adaptation to the host, especially during infection, are processes that are often linked in pathogenic bacteria. The human pathogen Staphylococcus aureus is equipped with a large arsenal of immune-modulating factors, allowing it to either subvert the host immune response or to create permissive niches for its survival. Recently, we showed that one of the low-molecular-weight protein tyrosine phosphatases produced by S. aureus, PtpA, is secreted during growth. Here, we report that deletion of ptpA in S. aureus affects intramacrophage survival and infectivity. We also observed that PtpA is secreted during macrophage infection. Immunoprecipitation assays identified several host proteins as putative intracellular binding partners for PtpA, including coronin-1A, a cytoskeleton-associated protein that is implicated in a variety of cellular processes. Of note, we demonstrated that coronin-1A is phosphorylated on tyrosine residues upon S. aureus infection and that its phosphorylation profile is linked to PtpA expression. Our results confirm that PtpA has a critical role during infection as a bacterial effector protein that counteracts host defenses.