Impact of subinhibitory concentrations of metronidazole on proteome of Clostridioides difficile strains with different levels of susceptibility

Clostridioides difficile is responsible for various intestinal symptoms from mild diarrhea to severe pseudomembranous colitis and is the primary cause of antibiotic-associated diarrhea in adults. Metronidazole was the first-line treatment for mild to moderate C. difficile infections for 30 years. However, clinical failure and recurrence rates of metronidazole is superior to oral vancomycin and metronidazole is now recommended only as an alternative to vancomycin or fidaxomicin, for an initial non-severe infection. The mechanisms of treatment failure and infection recurrence remain unclear. Given the poor fecal concentrations of metronidazole, the bacteria may be exposed to subinhibitory concentrations of metronidazole and develop adaptation strategy, which is likely to be the origin of an increase in treatment failures. In this study, a proteomic approach was used to analyze changes in the proteome of two strains with different levels of susceptibility to metronidazole in the presence of subinhibitory concentrations of this antibiotic. The two strains were grown to stationary phase: CD17-146, a clinical C. difficile isolate with reduced susceptibility to metronidazole, and VPI 10463, a metronidazole susceptible strain. Our study revealed that, whatever the strain, subinhibitory concentrations of metronidazole modified the amount of proteins involved in protein biosynthesis, glycolysis, and protection against stress induced by metronidazole, as well as in DNA repair. Several proteins involved in stress response are known to be synthesized under the control of Sigma factor B, which suggests a close link between Sigma factor B and metronidazole. Interestingly, impact of metronidazole on protein production for VPI 10463 strain differed from CD17-146 strain, for which the amount of two proteins involved in biofilm formation of CD17-146 were modified by metronidazole.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils

ABSTRACT Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus. We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB. Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.

The Staphylococcus aureus AirSR Two-Component System Mediates Reactive Oxygen Species Resistance via Transcriptional Regulation of Staphyloxanthin Production

ABSTRACT Staphylococcus aureus is an important opportunistic pathogen and is the etiological agent of many hospital- and community-acquired infections. The golden pigment, staphyloxanthin, of S. aureus colonies distinguishes it from other staphylococci and related Gram-positive cocci. Staphyloxanthin is the product of a series of biosynthetic steps that produce a unique membrane-embedded C30 golden carotenoid and is an important antioxidant. We observed that a strain with an inducible airR overexpression cassette had noticeably increased staphyloxanthin production compared to the wild-type strain under aerobic culturing conditions. Further analysis revealed that depletion or overproduction of the AirR response regulator resulted in a corresponding decrease or increase in staphyloxanthin production and susceptibility to killing by hydrogen peroxide, respectively. Furthermore, the genetic elimination of staphyloxanthin during AirR overproduction abolished the protective phenotype of increased staphyloxanthin production in a whole-blood survival assay. Promoter reporter and gel shift assays determined that the AirR response regulator is a direct positive regulator of the staphyloxanthin-biosynthetic operon, crtOPQMN, but is epistatic to alternative sigma factor B. Taken together, these data indicate that AirSR positively regulates the staphyloxanthin-biosynthetic operon crtOPQMN, promoting survival of S. aureus in the presence of oxidants.

Modulation of Staphylococcus aureus Biofilm Matrix by Subinhibitory Concentrations of Clindamycin

ABSTRACTStaphylococcus aureusbiofilms are extremely difficult to treat. They provide a protected niche for the bacteria, rendering them highly recalcitrant toward host defenses as well as antibiotic treatment. Bacteria within a biofilm are shielded from the immune system by the formation of an extracellular polymeric matrix, composed of polysaccharides, extracellular DNA (eDNA), and proteins. Many antibiotics do not readily penetrate biofilms, resulting in the presence of subinhibitory concentrations of antibiotics. Here, we show that subinhibitory concentrations of clindamycin triggered a transcriptional stress response inS. aureusvia the alternative sigma factor B (σB) and upregulated the expression of the major biofilm-associated genesatlA,lrgA,agrA, thepsmgenes,fnbA, andfnbB. Our data suggest that subinhibitory concentrations of clindamycin alter the ability ofS. aureusto form biofilms and shift the composition of the biofilm matrix toward higher eDNA content. An understanding of the molecular mechanisms underlying biofilm assembly and dispersal in response to subinhibitory concentrations of clinically relevant antibiotics such as clindamycin is critical to further optimize antibiotic treatment strategies of biofilm-associatedS. aureusinfections.

Staphopains Modulate Staphylococcus aureus Biofilm Integrity

ABSTRACTStaphylococcus aureusis a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. TheS. aureusgenome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigBmutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that ansspB scpAdouble mutant restored ΔsigBbiofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment ofS. aureusbiofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved acrossS. aureusstrain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulatingS. aureusbiofilm architecture.