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

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.