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Title: Competition of staphylococci as probe for novel antibiotics
Author: Ghabban, H. A.
ISNI:       0000 0004 8505 7128
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Staphylococci are frequent members of the human skin microbiome. It was previously determined that Staphylococcus aureus meets widespread antagonism from coagulase-negative staphylococci. Furthermore, cumulative antagonisms by species in the niche correlate with the absence of S. aureus nasal colonisation, supporting collective antagonism trait expression as a barrier. However, genetics factors and adaptive pathways of bacterial interactions remain uncharacterised. Using a two species system, strongly antagonistic inhibitor-producing S. epidermidis B155 was co-cultured with S. aureus SH1000 to identify determinants of competition and resistance using an experimental evolution approach. In the presence of S. epidermidis B155, S. aureus evolved a high relative fitness due to inhibitor resistance after several days of S. epidermidis co-culture. Unexpectedly, rather than known twocomponent system TCS loci associated with antimicrobial resistance, Illumina platform genome resequencing of S. aureus identified single nucleotide polymorphisms (SNPs) in both sensor kinase (desK) and response regulator (desR) genes that comprise the poorly described DesKR homologous TCS. The DesKR-like TCS system is located in an operon with two genes encoding an ABC transporter with homology to ABC polyketide efflux transporters. Genetic analysis identified that allelic replacement of desK or in-frame deletion of the SAOUHSC_01312 ABC efflux transporter gene decreased survival of S. aureus in competition with S. epidermidis, supporting the transporter as a resistance module. The desK gene mutant revealed reduced growth rate at both low (25°C) and high (42°C) temperatures compared with its parent strain. Gene inactivation of the desKR locus reduced pigment production with both desK and SAOUHSC_01312 mutants expressing less staphyloxanthin. PacBio genome sequencing of S. epidermidis B155 and antiSMASH identified a 55 kb plasmid with a unique polyketide/nonribosomal peptide synthetase (NRPS) gene cluster likely encoding the inhibitor of S. aureus growth. This cluster shares considerable sequence homology with a gene cluster encoding a polyketide/NRPS of Streptococcus mutans. Due to the production of this unique antimicrobial, further skin-isolated bacteria were screened for those that inhibit S. aureus SH1000 WT strain but not a desR SNP variant, thereby identifying Bacillus flexus B003. PacBio genome sequencing and antiSMASH identified genome-encoded polyketide synthase and linear azol(in)econtaining peptides (LAP) genes that might have the same mode of action as S. epidermidis B155 or DesKR resistance mechanism. RNAseq was performed to probe the regulon of the S. aureus DesKR-like TCS using WT to compare with the desR SNP variant, plus desK allelic replacement and SAOUHSC_01312 mutants. From this analysis, it was identified that the five genes SAOUHSC_01311-01312-desKR operon and SAOUHSC_01315 were very highly expressed in the desR SNP variant and these five genes are likely to comprise the DesKR-like regulon. Moreover, these data support the central role of this TCS in S. aureus resistance to the S. epidermidis B155 and B. flexus B003 antimicrobials. In contrast, transcriptome analysis of desK and transporter SAOUHSC_01312 mutant strains revealed hundreds of genes were differentially expressed in pathways associated with metabolism, virulence and regulations. The DesKR-like regulon has undefined roles that could be linked to membrane homeostasis and these findings provide significant insights into pathways that impact bacterial growth, resistance and microbial interaction. Ultimately, the TCS and its functions identified here might represent a future target for novel therapeutics targeting S. aureus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral