The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence-associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild-type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

Nudix proteins catalyze the hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. The genome of an important opportunistic human pathogen, Pseudomonas aeruginosa , encodes multiple Nudix proteins. To determine the role of nine Nudix hydrolases of the P. aeruginosa PAO1161 strain in its fitness, virulence or antibiotic resistance mutants devoid of individual enzymes were constructed and analyzed for growth rate, motility, biofilm formation, pyocyanin production, and susceptibility to oxidative stress and different antibiotics. The potential effect on bacterial virulence was studied using the C aenorhabditis elegans – P. aeruginosa infection model. Of the nine mutants tested, five had an altered phenotype in comparison with the wild‐type strain. The ΔPA3470, ΔPA3754, and ΔPA4400 mutants showed increased pyocyanin production, were more resistant to the β‐lactam antibiotic piperacillin, and were more sensitive to killing by H 2 O 2 . In addition, ΔPA4400 and ΔPA5176 had impaired swarming motility and were less virulent for C. elegans . The ΔPA4841 had an increased sensitivity to oxidative stress. These changes were reversed by providing the respective nudix gene in trans indicating that the observed phenotype alterations were indeed due to the lack of the particular Nudix protein.

Nudix proteins catalyse hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. Their widespread presence and broad substrate specificity suggest that they have important cellular functions. In this review, we summarize the state of knowledge on microbial Nudix proteins involved in pathogenesis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low‐oxygen activated ( lxa ) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa ‐encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56‐2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild‐type and complement strains but not the Δ usp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

For nearly half of the proteome of an important pathogen, Pseudomonas aeruginosa, the function has not yet been recognised. Here, we characterise one such mysterious protein PA2504, originally isolated by us as a sole partner of the RppH RNA hydrolase involved in transcription regulation of multiple genes. This study aims at elucidating details of PA2504 function and discussing its implications for bacterial biology. We show that PA2504 forms homodimers and is evenly distributed in the cytoplasm of bacterial cells. Molecular modelling identified the presence of a Tudor-like domain in PA2504. Transcriptomic analysis of a ΔPA2504 mutant showed that 42 transcripts, mainly coding for proteins involved in sulphur metabolism, were affected by the lack of PA2504. In vivo crosslinking of cellular proteins in the exponential and stationary phase of growth revealed several polypeptides that bound to PA2504 exclusively in the stationary phase. Mass spectrometry analysis identified them as the 30S ribosomal protein S4, the translation elongation factor TufA, and the global response regulator GacA. These results indicate that PA2504 may function as a tether for several important cellular factors.