Redox Imbalance Underlies the Fitness Defect Associated with Inactivation of the Pta-AckA Pathway in Staphylococcus aureus.
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ABSTRACT:
SUBMITTER: Marshall DD
PROVIDER: S-EPMC4875753 | biostudies-literature | 2016 Apr
REPOSITORIES: biostudies-literature
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Redox Imbalance Underlies the Fitness Defect Associated with Inactivation of the Pta-AckA Pathway in Staphylococcus aureus.
Marshall Darrell D DD Sadykov Marat R MR Thomas Vinai C VC Bayles Kenneth W KW Powers Robert R
Journal of proteome research 20160324 4
The phosphotransacetylase-acetate kinase (Pta-AckA) pathway is thought to be a vital ATP generating pathway for Staphylococcus aureus. Disruption of the Pta-AckA pathway during overflow metabolism causes significant reduction in growth rate and viability, albeit not due to intracellular ATP depletion. Here, we demonstrate that toxicity associated with inactivation of the Pta-AckA pathway resulted from an altered intracellular redox environment. Growth of the pta and ackA mutants under anaerobic ...[more]
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Project description:Staphylococcus aureus is an opportunistic pathogen with a clinical spectrum ranging from asymptomatic skin colonization to invasive infections. While traditional antibiotic therapies can be effective against S. aureus, the increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. Photodynamic inactivation (PDI) is an innovative and promising alternative to antibiotics. While progress has been made in our understanding of the bacterial response to PDI, major gaps remain in our knowledge of PDI tolerance, the global cellular response, and adaptive genomic mutations acquired as a result of PDI. To address these gaps, S. aureus HG003 and isogenic mutants with mutations in agr, mutS, mutL, and mutY exposed to single or multiple doses of PDI were assessed for survival and tolerance and examined by global transcriptome and genome analyses to identify regulatory and genetic adaptations that contribute to tolerance. Pathways in inorganic ion transport, oxidative response, DNA replication recombination and repair, and cell wall and membrane biogenesis were identified in a global cellular response to PDI. Tolerance to PDI was associated with superoxide dismutase and the S. aureus global methylhydroquinone (MHQ)-quinone transcriptome network. Genome analysis of PDI-tolerant HG003 identified a nonsynonymous mutation in the quinone binding domain of the transcriptional repressor QsrR, which mediates quinone sensing and oxidant response. Acquisition of a heritable QsrR mutation through repeated PDI treatment demonstrates selective adaption of S. aureus to PDI. PDI tolerance of a qsrR gene deletion in HG003 confirmed that QsrR regulates the S. aureus response to PDI.IMPORTANCE Staphylococcus aureus can cause disease at most body sites, with illness ranging from asymptomatic infection to death. The increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. S. aureus acquires resistance to antibiotics through multiple mechanisms, often by genetic variation that alters antimicrobial targets. Photodynamic inactivation (PDI), which employs a combination of a nontoxic dye and low-intensity visible light, is a promising alternative to antibiotics that effectively eradicates S. aureus in human infections when antibiotics are no longer effective. In this study, we demonstrate that repeated exposure to PDI results in resistance of S. aureus to further PDI treatment and identify the underlying bacterial mechanisms that contribute to resistance. This work supports further analysis of these mechanisms and refinement of this novel technology as an adjunctive treatment for S. aureus infections.
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Project description:Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids.IMPORTANCE Plasmids are major agents in the spread of antibiotic resistance genes among bacteria. How plasmids and their hosts coevolve to reduce the fitness cost associated with plasmid carriage when bacteria grow in an antibiotic-free environment is not well understood. Here, we investigated the cost and the genetic adaptations that occur during evolution in the absence of antibiotics when the bacterial pathogen Staphylococcus aureus acquires a new plasmid. Our results show the occurrence, at the end of evolution, of plasmid rearrangements mediated by insertion sequences that lead to the loss of antimicrobial resistance genes from the plasmid and an alleviated fitness cost. Our results thus highlight the probable benefits of reducing the use of antibiotics in management programs for the selection of S. aureus clones carrying plasmids that no longer confer resistance.
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Project description:BackgroundStaphylococcus aureus spreads its infections through biofilms. This usually happens in the stationary phase of S. aureus growth where it utilizes accumulated acetate as a carbon source via the phosphotrans-acetylase-acetate kinase (Pta-Ack) pathway. In which acetate kinase (ackA) catalyzes the substrate-level phosphorylation, a vital secondary energy-yielding pathway that promotes biofilms formation aids bacterium survival in hostile environments. In this study, we describe the cloning, sequencing, and expression of S. aureus ackA gene. The expression analysis of ackA gene in methicillin-resistant strains of S. aureus (MRSA) correlates with ackA activity and biofilm units. The uniqueness of ackA was analyzed by using in silico methods.ResultsElevated ackA gene expression was observed in MRSA strains, which correlates with increased ackA activity and biofilm units, explaining ackA role in MRSA growth and pathogenicity. The pure recombinant acetate kinase showed a molecular weight of 44 kDa, with enzyme activity of 3.35 ± 0.05 μM/ml/min. The presence of ACKA-1, ACKA-2 sites, one ATP, and five serine/threonine-protein kinase sites in the ackA gene (KC954623.1) indicated that acetyl phosphate production is strongly controlled. The comparative structural analysis of S. aureus ackA with ackA structures of Mycobacterium avium (3P4I) and Salmonella typhimurium (3SLC) exhibited variations as indicated by the RMSD values 1.877 Å and 2.141 Å respectively, explaining why ackA functions are differently placed in bacteria, concurring its involvement in S. aureus pathogenesis.ConclusionsOverall findings of this study highlight the correlation of ackA expression profoundly increases survival capacity through biofilm formation, which is a pathogenic factor in MRSA and plays a pivotal role in infection spreading.
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