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.

Transposon Insertion Site Sequencing of Providencia stuartii: Essential Genes, Fitness Factors for Catheter-Associated Urinary Tract Infection, and the Impact of Polymicrobial Infection on Fitness Requirements

“…It is also possible that some of the mutants might exhibit significant defects only when dramatically underrepresented in the input inoculum, such as in the case of the ϳ1:10,000 ratio that would have been present during the Tn-Seq screen but not during a 1:1 cochallenge. Regardless of the source of discrepancy, the validation rate resulting from combined assessments of mutants during single-species and polymicrobial infection was 9/12 (75%), which is consistent with the validation rates reported in other Tn-Seq studies (ranging from 75% to 86%) (27,29,(48)(49)(50)(51).…”

“…The mutant did not exhibit a fitness defect in the spleen, indicating that flagella do not contribute to development of bacteremia or survival once within the bloodstream. Flagella similarly do not contribute to Proteus mirabilis survival in the bloodstream (29). Contribution of twin arginine translocation to P. stuartii fitness during singlespecies and polymicrobial CAUTI.…”

“…Four mice per infection type per input pool were chosen for sequencing based on colonization levels of P. stuartii and high-quality DNA during sample preparation. A fitness index was calculated for each gene as previously described (27,29). P. stuartii fitness factors during single-species infection.…”

“…The tatC mutant exhibited significant defects during coinfection with P. mirabilis, confirming the importance of Tat substrates during both single-species infection and polymicrobial infection. The Tat system was also identified as a core fitness factor in P. mirabilis (27) and is critical for survival in the bloodstream (29). Thus, Tat substrates may be critical for the fitness of a variety of bacterial species within the urinary tract and bloodstream.…”

“…The chromosome and plasmid sequences of P. stuartii BE2467 (NCBI accession numbers CP017054, CP017055, and CP017056) (23) were combined into a single genome sequence with 1,000 N's added between each pair of contigs, and the coordinates of genes were adjusted accordingly. The Goodman In-Seq pipeline (63) was applied on the raw reads to perform read filtration, transposon nucleotide removal, debarcoding, alignment, and insertion calling, following a script that was previously used to map insertions onto the P. mirabilis genome (27,29).…”