Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia (PB) represents an important subset of S. aureus endovascular infections. In this study, we investigated potential genetic ...mechanisms underlying the persistent outcomes. Compared with resolving bacteremia (RB) isolates (defined as isolates associated with negative results of blood cultures 2-4 days after initiation of therapy), PB strains (defined as isolates associated with positive results of blood cultures ≥7 days after initiation of therapy) had significantly earlier onset activation of key virulence regulons and structural genes (eg, sigB, sarA, sae, and cap5), higher expression of purine biosynthesis genes (eg, purF), and faster growth rates, with earlier entrance into stationary phase. Importantly, an isogenic strain set featuring a wild-type MRSA isolate, a purF mutant strain, and a purF-complemented strain and use of strategic purine biosynthesis inhibitors implicated a causal relationship between purine biosynthesis and the in vivo persistent outcomes. These observations suggest that purine biosynthesis plays a key role in the outcome of PB and may represent a new target for enhanced efficacy in treating life-threatening MRSA infections.
The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate ...downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and ΔEL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ΔEL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ΔEL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1- and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ΔgraS and ΔEL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.
Staphylococcus aureus is the most common cause of life-threatening endovascular infections, including infective endocarditis (IE). These infections, especially when caused by methicillin-resistant ...strains (MRSA), feature limited therapeutic options and high morbidity and mortality rates.
Herein, we investigated the role of the purine biosynthesis repressor, PurR, in virulence factor expression and vancomycin (VAN) treatment outcomes in experimental IE due to MRSA.
The PurR-mediated repression of purine biosynthesis was confirmed by enhanced purF expression and production of an intermediate purine metabolite in purR mutant strain. In addition, enhanced expression of the transcriptional regulators, sigB and sarA, and their key downstream virulence genes (eg, fnbA, and hla) was demonstrated in the purR mutant in vitro and within infected cardiac vegetations. Furthermore, purR deficiency enhanced fnbA/fnbB transcription, translating to increased fibronectin adhesion versus the wild type and purR-complemented strains. Notably, the purR mutant was refractory to significant reduction in target tissues MRSA burden following VAN treatment in the IE model.
These findings suggest that the purine biosynthetic pathway intersects the coordination of virulence factor expression and in vivo persistence during VAN treatment, and may represent an avenue for novel antimicrobial development targeting MRSA.
Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth CAMHB) with bicarbonate (NaHCO
) increases β-lactam susceptibility of selected methicillin-resistant
(MRSA) strains ...("NaHCO
responsive"). This "sensitization" phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO
-mediated β-lactam MRSA sensitization using an
pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting
NaHCO
-responsive or NaHCO
-nonresponsive phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB with or without NaHCO
Intra-SEV MRSA killing was determined over 72-h exposures. In both "responsive" strains, supplementation with 25 mM or 44 mM NaHCO
significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤4 mg/liter) and resulted in bactericidal activity (≥3-log killing) in the model for both OXA and CFZ. In contrast, neither
-defined nonresponsive MRSA strain showed significant sensitization in the SEV model to either β-lactam. At both NaHCO
concentrations, the fractional time above MIC was >50% for both CFZ and OXA in the responsive MRSA strains. Also, in media containing RPMI plus 10% Luria-Bertani broth (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO
), both CFZ and OXA exhibited enhanced bactericidal activity against NaHCO
-responsive strains in the SEV model. Neither CFZ nor OXA exposures selected for emergence of high-level β-lactam-resistant mutants within SEVs. Thus, in this
model of endocarditis, in the presence of NaHCO
supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO
-supplemented media
.
Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop ...(EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 hNP-1), cutaneous β-defensin (human β-defensin 2 hBD-2), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.
Bacteremia is a life-threating syndrome often caused by methicillin-resistant Staphylococcus aureus (MRSA). Thus, there is an urgent need to develop novel approaches to successfully treat this ...infection. Staphylococcal accessory regulator A (SarA), a global virulence regulator, plays a critical role in pathogenesis and β-lactam antibiotic resistance in Staphylococcus aureus. Hypericin is believed to act as an antibiotic, antidepressant, antiviral and non-specific kinase inhibitor. In the current study, we investigated the impact of hypericin on β-lactam antibiotics susceptibility and mechanism(s) of its activity. We demonstrated that hypericin significantly decreased the minimum inhibitory concentrations of β-lactam antibiotics (e.g., oxacillin, cefazolin and nafcillin), biofilm formation and fibronectin binding in MRSA strain JE2. In addition, hypericin significantly reduced sarA expression, and subsequently decreased mecA, and virulence-related regulators (e.g., agr RNAⅢ) and genes (e.g., fnbA and hla) expression in the studied MRSA strain. Importantly, the in vitro synergistic effect of hypericin with β-lactam antibiotic (e.g., oxacillin) translated into in vivo therapeutic outcome in a murine MRSA bacteremia model. These findings suggest that hypericin plays an important role in abrogation of β-lactam resistance against MRSA through sarA inhibition, and may allow us to repurpose the use of β-lactam antibiotics, which are normally ineffective in the treatment of MRSA infections (e.g., oxacillin).
In this study, the authors demonstrated that hypericin, as a sarA inhibitor, had synergistic activity with β-lactams both in vitro and in a murine bacteremia model due to MRSA. The mechanisms may be related to significantly reduced biofilm formation, fibronectin binding and expression of virulence genes (e.g., fnbA and hla). Display omitted
Methicillin-resistant
(MRSA) poses significant therapeutic challenges related to its frequency in clinical infections, innate virulence properties, and propensity for multiantibiotic resistance. MRSA ...is among the most common causes of endovascular infections, including infective endocarditis (IE). Our objective was to employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE. TTE was utilized to evaluate the
effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (versus daptomycin alone). Primary intravegetation outcomes were maximum size, weights at sacrifice, and MRSA counts at infection baseline versus after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment day 1). A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase plus daptomycin versus the daptomycin alone therapy groups (
= 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower than those of the daptomycin alone group (
< 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (
= 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower than those of untreated controls (
< 0.0001) and the daptomycin alone group (
< 0.0001). This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size, and weight. Moreover, TTE appears to be an efficient noninvasive tool to assess therapeutic efficacies in experimental MRSA IE.
Using isogenic clinical bloodstream Staphylococcus aureus strains from a patient with relapsing endocarditis, we investigated the transcriptional profiles of the mprF and dlt genes in the context of ...cell-surface charge and daptomycin nonsusceptibility. As in prior studies, a point mutation within mprF was observed in the daptomycin-nonsusceptible strain. However, neither the transcriptional profile of mprF nor the membrane phospholipid analyses were compatible with the anticipated mprF gain-in-function phenotype. In contrast, we demonstrated enhanced dlt expression coincident with increased positive surface charge and reduced daptomycin binding
Invasive methicillin-resistant
(MRSA) infections are leading causes of morbidity and mortality that are complicated by increasing resistance to conventional antibiotics. Thus, minimizing virulence ...and enhancing antibiotic efficacy against MRSA is a public health imperative. We originally demonstrated that diflunisal (DIF; 2-hydroxy-5-(2,4-difluorophenyl) benzoic acid) inhibits
virulence factor expression. To investigate pharmacophores that are active in this function, we evaluated a library of structural analogues for their efficacy to modulate virulence phenotypes in a panel of clinically relevant
isolates in vitro. Overall, the positions of the phenyl, hydroxyl, and carboxylic moieties and the presence or type of halogen (F vs. Cl) influenced the efficacy of compounds in suppressing hemolysis, proteolysis, and biofilm virulence phenotypes. Analogues lacking halogens inhibited proteolysis to an extent similar to DIF but were ineffective at reducing hemolysis or biofilm production. In contrast, most analogues lacking the hydroxyl or carboxylic acid groups did not suppress proteolysis but did mitigate hemolysis and biofilm production to an extent similar to DIF. Interestingly, chirality and the substitution of fluorine with chlorine resulted in a differential reduction in virulence phenotypes. Together, this pattern of data suggests virulence-suppressing pharmacophores of DIF and structural analogues integrate halogen, hydroxyl, and carboxylic acid moiety stereochemistry. The anti-virulence effects of DIF were achieved using concentrations that are safe in humans, do not impair platelet antimicrobial functions, do not affect
growth, and do not alter the efficacy of conventional antibiotics. These results offer proof of concept for using novel anti-virulence strategies as adjuvants to antibiotic therapy to address the challenge of MRSA infection.
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