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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.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals is largely attributed to its virulence factor repertoire and the sophisticated regulatory network controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu, and to withstand Triton X-100 induced autolysis. SA564 ΔptpB mutant cells were additionally ingested faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB contributes by several ways positively to the ability of S. aureus to evade host innate immunity.

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.

Purpose The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro. Methods Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli (E. coli) plates and observed for 3 weeks. Results All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again. Conclusions The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E. coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents. Translational relevance Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E. coli agar plate assay.

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.