Regulatory and Metabolic Networks for the Adaptation of Pseudomonas aeruginosa Biofilms to Urinary Tract-Like Conditions

Abstract

Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.

A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

“…Our results are in good agreement with the findings of Tielen et al who showed that P. aeruginosa PAO1 is not able to grow on urea as a sole carbon source in AUM (21). Various secreted or surface-exposed virulence factors of P. aeruginosa could be involved in immune evasion, that is, elastase (LasB), alkaline metalloprotease (AprA), insulin cleaving membrane proteinase (ICMP), elongation factor Tu (EF-Tu), as well as in iron acquisition, that is, PvdS-regulated endoprotease (PrpL) and siderophores.…”

“…6) and taken up by transporters such as an amino acid ABC transporter, which appeared to be highly abundant in the catheter biofilm (Table I). This hypothesis is supported by the finding that P. aeruginosa PAO1 is able to grow in AUM with peptone as the sole carbon source (21).…”

“…The mentioned studies identified the lack of freely available trace metals, especially iron, as a major limiting factor in human (and murine) urine during urinary tract infections (UTIs). For uropathogenic P. aeruginosa a strong iron limitation response was observed when cultured as colony biofilm for 6 days on artificial urine medium (AUM) agar (21). Moreover, fimbrial genes, like mrp and fim, were found highly expressed in uropathogenic P. mirabilis and E. coli (18), highlighting the importance of bacterial adherence during CAUTIs.…”

Gene Regulatory and Metabolic Adaptation Processes of Dinoroseobacter shibae DFL12T during Oxygen Depletion

“…Correspondingly, NorCBQ and NosZY proteins were detected 30 -60 min after the shift (supplemental Table S4). This abundance pattern suggests a fine-tuned regulatory network with (i) nitrite formed by Nap most likely triggering nir gene expression and (ii) the nitrite reduction product NO initiating expression of nor and nos genes as described for P. aeroginosa (30). Furthermore, direct reduction of nitrite prevents its intermediary accumulation, thereby avoiding accumulation of toxic nitrite levels.…”

“…Pairwise comparisons of obtained expression rates for different time points after the shift to anoxic conditions with data obtained prior to anaerobic shift (0 min) were made ( Table 1). The investigated time points were 5,10,15,20,30,60, and 120 min after the oxygen supply was switched off (Fig. 1).…”