Dissecting the lifestyle of Klebsiella pneumoniae and Vibrio cholerae using chemical genomics

Alao, Micheal Bukola (2025). Dissecting the lifestyle of Klebsiella pneumoniae and Vibrio cholerae using chemical genomics. University of Birmingham. Ph.D.

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Abstract

Klebsiella pneumoniae and Vibrio cholerae are important human pathogens with increasing antimicrobial resistance in the clinic. Although, genome sequencing information are available for both strains, their genomes are still poorly annotated. This hinders our better understanding of their lifestyles, which is important in discovering new drug targets and tackling antimicrobial resistance. In this thesis, we applied chemical genomics to improve functional annotations of the genomes of both organisms. To do this, we systematically assessed the fitness of ordered single gene deletion transposon mutant libraries of K. pneumoniae (4346 mutants) and V. cholerae (3060 mutants) against ~200 chemical and environment perturbations (stresses) targeting different cellular processes. The resulting stress-response datasets showed high replicate reproducibility with correlation coefficient greater than 0.7 in all experiments and benchmarking against known biology of functionally related genes having similar responses across all the stresses indicated that our datasets are of good quality. The responsive genomes, which are genes inactivated in mutants with significant responses to a stress, were 42% and 52% of the genome of K. pneumoniae and V. cholerae respectively. More so, we identified 348 and 240 genes essential for growth in different stress conditions in K. pneumoniae and V. cholerae respectively.

Additionally, functions were predicted to hundreds of hypothetical genes with the functions further validated via computational methods using Alpha Fold, ChimeraX and Dali. For example, KPNIH1_22370 was predicted to be a membrane protein with its mutant showing reduced fitness in triton-x and correlation coefficient of 0.64 with yidD. Then, possible interactions were validated between KPNIH1_22370 and yidD on ChimeraX. Similarly, VC_0032 was predicted to be involved in carbon starvation with its mutant showing reduced fitness in minimal carbon stresses and correlation coefficient of 0.79 with vcc collagenase. Then, possible interactions between VC_0032 and vcc collagenase were validated on ChimeraX and Dali. Furthermore, in K. pneumoniae, single mutants of envC and hflC genes showed reduced fitness in aminoglycosides while single mutants of nuo, cyo and sdh genes showed increased fitness in aminoglycosides. This suggests that with leaky membrane from the inactivation of envC and hflC, more aminoglycosides get into the cell and inhibit protein synthesis. Additionally, inactivation of nuo, cyo and sdh genes resulting in increased fitness suggests there is a reduction in aminoglycosides uptake in dysfunctional electron transport chain. More so, mutants of tcpP showed reduced fitness in carbon starvation and acidic condition in V. cholerae. This suggests that tcpP plays an important role in the survival of V. cholerae in aquatic environments and in human host.

In addition, our small-scale screens showed that chemical genomics is applicable in screening few strains against few conditions. In this thesis, we evaluated the fitness of six mutants of beta barrel assembly machinery (Bam) associated proteins in 28 unique stress conditions to show that the Bam associated proteins do not phenocopy each other. Similarly, we assessed the fitness of 34 single and double mutants of peptidoglycan (PG) enzymes in nine unique envelope stresses to show that PBP1B, encoded as mrcB, is important for fitness in salt stresses. More so, chemical genomics was adapted in evaluating the minimum inhibitory concentrations (MIC) of 96 clinical isolates in chlorhexidine with isolates of K. pneumoniae with the highest MIC. In conclusion, our stress-response datasets from evaluating the fitness of the strains in the stresses are rich in information that helped us to better understand the biology of the organisms.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):