Nucleotide Biosynthesis Is Critical for Growth of Bacteria in Human Blood

Abstract

Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another Gram-negative pathogen, Salmonella enterica, and in the Gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections.

Genome-Wide Screening with Hydroxyurea Reveals a Link between Nonessential Ribosomal Proteins and Reactive Oxygen Species Production

“…The Keio Collection has been used by many groups to observe the effects of gene deletions under specific conditions of interest. These studies have included the determination of mutations affecting antibiotic hypersensitivity (2,3), swarming motility (4), biofilm formation (5), growth in human blood (6), recipient ability in plasmid conjugation (7), cysteine tolerance and production (8), colicin import and cytotoxicity (9), de-ethylation of 7-ethoxycoumarin (10), and glycogen metabolism (11). In most of these studies, the sets of relevant gene deletions show enrichment for specific cellular functions linked to the conditions of the screen.…”

Identification of Streptococcus sanguinis Genes Required for Biofilm Formation and Examination of Their Role in Endocarditis Virulence

“…It is also interesting that the S. sanguinis purB mutant is attenuated in both biofilm formation and virulence for endocarditis whereas the purL mutant exhibits only biofilm reduction. Both the purB gene and the purL gene have been shown to be important for virulence in studies performed with S. pneumoniae (38) and with Bacillus anthracis (65). Given these results and the overall similarity of the S. pneumoniae and S. sanguinis purine pathways (P. Xu, unpublished observations), it is more surprising that purL is not required for virulence in S. sanguinis than that purB is.…”

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

“…Interestingly, this rate-limiting DNA biosynthesis enzyme has a 56601-specific missense mutation causing an A703T substitution within a barrel domain. It was previously reported that, compared with other metabolic pathways, inactivation of purine/pyrimidine pathway genes in Escherichia coli and some other bacteria was the key factor for limiting their growth in human serum [25]. Therefore, these variations are likely, to a certain extent, to contribute to the avirulence property of strain IPAV.…”