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Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection.

ABSTRACT: The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality.

SUBMITTER: Armbruster CE

PROVIDER: S-EPMC6497324 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:Klebsiella pneumoniae and Proteus mirabilis form mixed biofilms in catheter-associated urinary tract infections. However, co-inoculation of P. mirabilis with K. pneumoniae in artificial urine medium (AUM) resulted in a drastic reduction of K. pneumoniae cells in both biofilm and planktonic growth. Here, the mechanism behind this competitive interaction was studied. Both pH and aqueous ammonia (NH3aq) increased in mixed cultures (to 9.3 and 150 mM, respectively), while K. pneumoniae viable cells dramatically diminished over time (>6-log reduction, p < 0.05). Mixed cultures developed in either 2-(N-morpholino) ethanesulfonic acid (MES)-buffered AUM (pH 6.5) or AUM without urea did not show bacterial competition, evidencing that the increase in pH and/or NH3aq concentration play a role in the competitive interaction. Viability of K. pneumoniae single-species cultures decreased 1.5-log in alkaline AUM containing 150 mM NH3aq after 24 h inoculation, suggesting that ammonia is involved in this inter-species competition. Besides NH3aq, additional antimicrobials should be present to get the whole competitive effect. Supernatants from P. mirabilis-containing cultures significantly diminished K. pneumoniae viability in planktonic cultures and affected biofilm biomass (p < 0.05). When subjected to evaporation, these supernatants lost their antimicrobial activity suggesting the volatile nature of the antimicrobial compounds. Exposure of K. pneumoniae to volatile compounds released by P. mirabilis significantly decreased cell viability in both planktonic and biofilm cultures (p < 0.05). The current investigation also evidenced a similar bactericidal effect of P. mirabilis volatiles over Escherichia coli and Morganella morganii. Altogether, these results evidence the secretion of ammonia and other volatile compounds by P. mirabilis, with antimicrobial activity against gram-negative uropathogens including K. pneumoniae. This investigation provides novel insight into competitive inter-species interactions that are mediated by production of volatile molecules.

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Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection.

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