Investigating the molecular basis of histidine catabolism in a human pathogenic bacterium Pseudomonas aeruginosa PAO1 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Auckland, New Zealand
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Date
2021
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Massey University
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Abstract
Pseudomonas aeruginosa is an opportunistic and a nosocomial pathogen of significant medical concern, particularly for cystic fibrosis patients. The extensive metabolic flexibility coupled with an array of virulence factors make them ubiquitous and successful in causing persistent multi-drug resistant infections. Pathogens exploit nutrient-rich hosts, and thus nutrients can be considered as signals perceived by bacteria that allow host recognition and coordination of expression of metabolic and virulence genes for successful colonization. A
deeper understanding of the metabolic pathways and host perception mechanisms are significant from a therapeutic perspective in the current era of antibiotic resistance.
Histidine is an amino acid that can serve as a source of carbon and nitrogen to many bacteria.
Histidine catabolism in Pseudomonas spp., is widely known to occur via a 5-step enzymatic pathway, and the genes for histidine utilization (hut) are negatively regulated by HutC protein. The enteric bacteria and some others utilize a 4-step enzymatic pathway for histidine catabolism, which differs from the 5-step pathway in the direct conversion of intermediate
formiminoglutamate (FIGLU) to glutamate. However, P. aeruginosa contains an additional operon (dislocated from the hut locus) encoding for formimidoylglutamase enzyme and its
regulator, which can break down FIGLU similar to 4-step pathway. Previous studies report the
accumulation of histidine metabolites, urocanate and FIGLU, in the mammalian tissues and
reduced virulence of P. aeruginosa defective in histidine catabolism towards animal models.
But the implications of the presence of two pathways for histidine catabolism or mechanisms associated with virulence remain elusive. This prompted us to examine the hut pathways and mechanisms that link hut with virulence in P. aeruginosa PAO1.
First, computational analysis identified a transporter gene (named figT) adjacent to
formimidoylglutamase enzyme (FigA) and transcriptional regulator FigR. This led to a new
hypothesis that the three genes (figRAT) are responsible for the uptake and utilization of
FIGLU, and they are not involved in histidine utilization as previously thought. Genetic
analyses utilizing site-directed mutagenesis and lacZ reporter fusions confirmed that figT
encodes for a FIGLU-specific transporter whose expression is induced by FIGLU. The figT
gene is co-transcribed with figRA, and its expression is activated by FigR. Furthermore, gene
expression studies indicate that FIGLU is the physiological inducer of fig operon, while
histidine and urocanate are indirect inducers (by virtue of conversion to FIGLU). Growth and
fitness assays revealed that histidine is predominantly catabolised via the 5-step hut pathway (not via the FigRAT system). Together, our genetic and phenotypic data show that fig operon
is involved in the direct utilization of FIGLU from the environment. Phylogenetic analysis showed that figRAT genes are highly conserved and present in all completely sequenced strains of P. aeruginosa, but we found no evidence for horizontal gene transfer events.
Previous work in Zhang’s laboratory suggests that urocanate derived from host tissues could serve as a signalling molecule, eliciting P. aeruginosa infections via interaction with the HutC regulator. Here, we aimed to seek further genetic, biochemical, and phenotypic evidence to improve our understanding of the global regulatory roles for HutC beyond histidine catabolism and determine their potential contribution to the colonization of eukaryotic hosts. Utilizing in silico analysis, we predicted 172 novel HutC-target sites in the genome of P. aeruginosa PAO1 with a P value less than 10-4. Six selected candidates were subject to experimental verification for HutC binding by means of gel shift assays (EMSA) and/or DNAse I footprinting assays, and all were able to bind with purified HutChis6 proteins. Further, a hutC deletion mutant was constructed by site-directed mutagenesis and subjected to phenotypic characterization. Phenotypic analyses revealed that hutC is involved in biofilm formation, tobramycin-induced biofilm formation, cell motility, and pyoverdine production. Significantly, we found that mutation of hutC resulted in reduced killing of C. elegans by P. aeruginosa PAO1.
Finally, we observed distinct binding patterns for HutC interaction with the hutF promoter DNAs in P. aeruginosa PAO1 and P. fluorescens SBW25 (a model plant-colonizing bacterium used for studies of histidine catabolism). Molecular investigations revealed that the differences were not caused by HutC proteins from either species, but HutC recognized a distinct site proximal to hutFSBW25. This site displayed sequence similarity with the NtrC-binding site and was called the Pntr site. Functional analysis of the significance of Pntr site identified that Pntr site is necessary for the wild-type level production of HutF in P. fluorescens SBW25 during growth on histidine.
Overall, the results from this study enhance our understanding of hut catabolism in Pseudomonas and contribute to novel molecular mechanisms associated with the virulence of P. aeruginosa PAO1. The identification of fig operon for the utilization of FIGLU (accumulated in host tissues) and global regulatory role of HutC in gene expression have broader implications from a therapeutic perspective in treating P. aeruginosa PAO1 infections. The ability of HutC to recognise multiple distinct DNA-binding sites suggests complex modes of gene regulation mediated by HutC and promotes further studies to fully understand the functional significance of genes in the HutC regulon.
Description
Figures 1.3, 1.4, 1.6, 1.7 & 1.8 (Oxford University Press, License ID 5424940483785) are re-used with permission.
Keywords
Amino acids, Metabolism, Bacterial genetics, Genetic regulation, Pseudomonas aeruginosa