Searching for Biological Function of the Mysterious PA2504 Protein from Pseudomonas aeruginosa.

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.

Nudix proteins affecting microbial pathogenesis.

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.

Nudix-type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa.

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.

A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis.

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 Δusp76 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.

Clinical Study of Oral Mucosal Lesions in the Elderly-Prevalence and Distribution.

BACKGROUND: The aim of the study was to determine the prevalence, pattern, and distribution of oral mucosa lesions in elderly patients attending an outpatient oral pathology clinic. METHODS: A retrospective analysis of 2747 patients' medical files was performed. Of these, 1398 (50.6%) belonged to seniors. The mean age was 69.8 ± 7.1, and women were in the majority. RESULTS: Among the entire group of patients, the three most common mucosal lesions were: oral lichenoid diseases (OLDs), lingual changes, and small nodules. In the female group, the most common diagnoses were: OLDs, lingual changes, and oral candidiasis; in the male population, the most common diagnoses were: lingual changes, small nodules, and OLDs. Those suffering from OLDs were the youngest patients, and those with focal oral mucosa erosions and ulcerations were the oldest patients. In the groups aged 60-70 and 71-80 years old, the most common diagnoses were OLDs, and in the oldest group (+80 years old), they were lingual lesions. CONCLUSIONS: Oral health has an obvious impact on the functional, psychological, and behavioral quality of life. There is a small number of publications concerning the prevalence and distribution of oral mucosa lesions in the senior population of European countries. Our study is filling in that void.

The Prevalence of Oral Mucosa Lesions in Pediatric Patients.

The prevalence of oral mucosa lesions (OMLs) among children varies from 4.1% up to 69.5%. There is a lack of sufficient epidemiological data and adequate knowledge about OMLs in relation to minors' gender and age. The aim of the study was to evaluate the prevalence of OMLs in children, patients of the oral pathology clinic in south-western parts of Poland, and to investigate the potential correlation between the occurrence of particular types of OMLs and the gender and age. A retrospective study was performed using a total of 2474 clinical charts from 2015 to 2019. Data collected included age, gender, and OMLs' type. The prevalence of OMLs in minors was 5.21%. Aphthae was the most frequent diagnosis. Boys were more commonly affected, and traumatic erosion and ulcers were significantly more often detected in males. The mean age of children was 8 y/o, preschoolers were significantly more often diagnosed with geographic tongue, while Morsicatio buccarum was significantly more common in school children and adolescents. Clinicians should be familiar with OMLs prevalence and with its specific frequency in connection to age and gender of children. Furthermore, they should be aware of the diversity of OMLs found in the oral cavity of children and that their frequency in the pediatric population is different from that in adults.

Individual Nudix hydrolases affect diverse features of Pseudomonas aeruginosa.

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 Caenorhabditis 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 ß-lactam antibiotic piperacillin, and were more sensitive to killing by H2 O2 . 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.

A Case of Lingua Villosa Nigra (Black Hairy Tongue) in a 3-Month-Old Infant.

BACKGROUND Black hairy tongue is a self-limiting, usually asymptomatic, benign lesion that most often affects men and people aged over 30-40 years. The lesion is extremely rare among newborns and infants. Lingua villosa nigra is characterized by the presence of elongated filiform papillae of the dorsum of the tongue, which gives a hairy appearance. The overgrown papillae can accumulate fungi, bacteria, pigments originating from food, or any other debris that can contribute to the discoloration of the tongue. The prognosis for black hairy tongue is positive. Gentle tongue brushing or scraping as well as the elimination of predisposing factors usually leads to cessation of the lesion. CASE REPORT A generally healthy, exclusively breastfed 3-month-old female baby was admitted to the Oral Pathology Department of Wroclaw Medical University due to a persistent tongue lesion. Intraorally, dark, blackish, elongated tongue papillae were observed. Three weeks earlier, the baby's pediatrician had diagnosed thrush and prescribed systemic antifungal treatment with Nystatinum, without prior mycological examination. The lesion did not resolve and the girl was referred to the Department of Oral Pathology. A meticulous medical and dietary interview revealed that since the 28th day of life the baby had been supplemented with vitamin C. This, together with an intraoral examination, led to the diagnosis of black hairy tongue. The lesion disappeared partially after 4 weeks of tongue brushing. CONCLUSIONS To prevent infants from undergoing persistent and unnecessary treatment (topical or systemic drugs) or additional diagnostic procedures, such as biopsy, it is essential to be familiar with the characteristics of lingua villosa nigra as well as its origin and management. The consideration of this condition is invaluable for babies' health and safety.