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Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa.

ABSTRACT: UNLABELLED:Fitness costs play a key role in the evolutionary dynamics of antibiotic resistance in bacteria by generating selection against resistance in the absence of antibiotics. Although the genetic basis of antibiotic resistance is well understood, the precise molecular mechanisms linking the genetic basis of resistance to its fitness cost remain poorly characterized. Here, we examine how the system-wide impacts of mutations in the RNA polymerase (RNAP) gene rpoB shape the fitness cost of rifampin resistance in Pseudomonas aeruginosa. Rifampin resistance mutations reduce transcriptional efficiency, and this explains 76% of the variation in fitness among rpoB mutants. The pleiotropic consequence of rpoB mutations is that mutants show altered relative transcript levels of essential genes. We find no evidence that global transcriptional responses have an impact on the fitness cost of rifampin resistance as revealed by transcriptome sequencing (RNA-Seq). Global changes in the transcriptional profiles of rpoB mutants compared to the transcriptional profile of the rifampin-sensitive ancestral strain are subtle, demonstrating that the transcriptional regulatory network of P. aeruginosa is robust to the decreased transcriptional efficiency associated with rpoB mutations. On a smaller scale, we find that rifampin resistance mutations increase the expression of RNAP due to decreased termination at an attenuator upstream from rpoB, and we argue that this helps to minimize the cost of rifampin resistance by buffering against reduced RNAP activity. In summary, our study shows that it is possible to dissect the molecular mechanisms underpinning variation in the cost of rifampin resistance and highlights the importance of genome-wide buffering of relative transcript levels in providing robustness against resistance mutations. IMPORTANCE:Antibiotic resistance mutations carry fitness costs. Relative to the characteristics of their antibiotic-sensitive ancestors, resistant mutants show reduced growth rates and competitive abilities. Fitness cost plays an important role in the evolution of antibiotic resistance in the absence of antibiotics; however, the molecular mechanisms underlying these fitness costs is not well understood. We applied a systems-level approach to dissect the molecular underpinnings of the fitness costs associated with rifampin resistance in P. aeruginosa and showed that most of the variation in fitness cost can be explained by the direct effect of resistance mutations on the enzymatic activity of the mutated gene. Pleiotropic changes in transcriptional profiles are subtle at a genome-wide scale, suggesting that the gene regulatory network of P. aeruginosa is robust in the face of the direct effects of resistance mutations.

SUBMITTER: Qi Q

PROVIDER: S-EPMC4324240 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa.

Qi Qin Q Preston Gail M GM MacLean R Craig RC

mBio 20141209 6

<h4>Unlabelled</h4>Fitness costs play a key role in the evolutionary dynamics of antibiotic resistance in bacteria by generating selection against resistance in the absence of antibiotics. Although the genetic basis of antibiotic resistance is well understood, the precise molecular mechanisms linking the genetic basis of resistance to its fitness cost remain poorly characterized. Here, we examine how the system-wide impacts of mutations in the RNA polymerase (RNAP) gene rpoB shape the fitness co ...[more]

PMID: 25491352

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Project description:The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa (P. aeruginosa) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU. It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy.IMPORTANCEThe bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy, involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.

Project description:BackgroundRiemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer.ResultsComparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10- 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h.ConclusionsMutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo.

Project description:The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

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Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa.

Publications

Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa.

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